Capping prone RNA polymerase enzymes and their applications

ABSTRACT

The invention provides a chimeric enzyme comprising at least one catalytic domain of a RNA triphosphatase, at least one catalytic domain of a guanylyltransferase, at least one catalytic domain of a N 7 -guanine methyltransferase, and at least one catalytic domain of a DNA-dependant RNA polymerase. The invention also provides pharmaceutical composition comprising said chimeric enzyme and uses of said chimeric enzyme.

The sequence listing text file (65002US_Sequence_Listing.txt, created Mar. 31, 2014, 163,840 bytes) is incorporated by reference in its entirety herein.

The present invention relates to the field of transgenesis, particularly in eukaryotic cells.

In particular, the invention relates to a chimeric enzyme useful for the production of RNA molecules with 5′-terminal m⁷GpppN cap structures.

Eukaryotic expression is very widely used in the life sciences, biotechnology and medicine. Thus, many methods for efficient transgenesis in eukaryotic cells have been developed. Common DNA sources and delivery mechanisms are viruses (e.g. baculovirus, retrovirus, or adenovirus) or non-viral vectors including plasmids and artificial chromosomes.

Because of their simplicity, the non-viral plasmids are commonly used as expression vectors for gene transfer into eukaryotic cells both in vitro and in vivo applications. However, the levels of transgene expression achieved by non-viral methods are usually modest and declines rapidly. A common explanation for this modest efficacy is the fact that DNA molecules, which are over approximately 40,000 Daltons, are too large to pass through the nuclear pores and enter the nucleus, where they are transcribed by the nuclear RNA polymerase II (Lang, Scholz et al. 1986; Zabner, Fasbender et al. 1995). In fact, only a very small amount (<0.1-0.001%) of large DNA molecules is actively transferred from the cytoplasm to the nucleus of eukaryotic cells. The mechanisms by which expression rapidly declines are also possibly nuclear-specific and related to the silencing of transgene expression by various epigenetic mechanisms (Loser, Jennings et al. 1998; Gill, Smyth et al. 2001; Miao, Thompson et al. 2001; Nicol, Wong et al. 2002; Miao, Ye et al. 2003).

Other drawbacks of transgenesis methods using endogenous RNA transcription system of eukaryotic cells also restrain their use. Firstly, the weak processivity of nuclear eukaryotic RNA polymerases (e.g. 10-20 nucleotides/second for RNA polymerase II) (Fire, Samuels et al. 1984; Ucker and Yamamoto 1984; Bengal, Flores et al. 1991; Izban and Luse 1992). Secondly, the competition between endogenous gene transcription and transgene transcription. Thirdly, the extreme complexity of eukaryotic RNA polymerases, which are made of several subunits (e.g. 12 subunits for RNA polymerase II and regulated by multiple transcription factors (Lodish, Berk et al. 2008).

In view of these disadvantages, some transgenesis methods based on bacteriophage DNA-dependent RNA polymerases have been developed. These methods have notably the advantage of not using the endogenous RNA transcription system of eukaryotic cells but some bacteriophage DNA-dependent RNA polymerases, which have a higher processivity than the eukaryotic RNA polymerases.

The pET expression system is a popular method for gene expression in prokaryotes (Studier, Rosenberg et al. 1990). It relies on the expression of the bacteriophage single-subunit T7 DNA-dependent RNA polymerase (T7 RNA polymerase, T7RNAP), the product of T7 gene 1, to transcribe genes of interest engineered to be expressed under the control of a T7 promoter. The pET expression system has been adapted to eukaryotic cells and is usually designated as the hybrid RNA polymerase. However, in an eukaryotic environment, the high enzymatic activity of the T7 DNA dependent RNA polymerase remarkably contrasts with very weak translation yields of the T7 transcripts (Fuerst, Niles et al. 1986). The absence of maturation of the transcripts in eukaryotic cells, which are neither modified by the addition of cap structures at their 5′-terminal (Benton, Eng et al. 1990; Dower and Rosbash 2002), nor strongly polyadenylated at their 3′-terminal (Mifflin and Kellems 1991; Dower and Rosbash 2002), provides an explanation for this discrepancy.

Methods for improving the translatability of uncapped transcripts produced by the hybrid system have thus been developed, like the vaccinia virus/bacteriophage RNAP hybrid expression system. This eukaryotic expression system is based on a recombinant vaccinia virus that synthesizes the bacteriophage T7 DNA dependent RNA polymerase in the cytoplasm of infected mammalian cells (Fuerst, Niles et al. 1986; Fuerst, Earl et al. 1987; Elroy-Stein, Fuerst et al. 1989; Fuerst, Fernandez et al. 1989; Fuerst and Moss 1989; Elroy-Stein and Moss 1990). The target gene for the bacteriophage RNA polymerase, flanked by T7 promoter and termination sequences, is introduced into infected cells either by transfection of a recombinant plasmid or by infection with a second recombinant vaccinia virus (Fuerst, Niles et al. 1986; Elroy-Stein, Fuerst et al. 1989; Elroy-Stein and Moss 1990). It was expected that the vaccinia virus-encoded cytoplasmic enzymes for mRNA capping would act on the T7 transcripts to improve their translatability. However, the capping of T7 transcripts remains infra-optimal (Fuerst and Moss 1989). For instance, using this expression system, it was found that T7 transcripts can comprise up to 30% of total cytoplasmic RNA after a 24 hour period, but only 5%-10% of T7 transcripts contained 5′-terminal cap structures (Fuerst and Moss 1989). Although rather efficient, technical drawbacks of the vaccinia virus/bacteriophage RNAP hybrid expression system clearly restrain its generalization and use at large-scale. Firstly, this system is based on recombinant vaccinia viruses, which are infectious for humans. Therefore, handling these recombinant viruses require specific laboratory facilities and practices. An attenuated avian host-range-restricted strain, i.e. the modified vaccinia Ankara (MVA), which aborts its replicative cycle at a late-stage packaging step in human cells, can be used to better control this hazard (Wyatt, Moss et al. 1995; Engleka, Lewis et al. 1998). Secondly, the recombinant vaccinia or MVA viruses are cytotoxic. Therefore, the vaccinia virus/bacteriophage RNAP hybrid expression system can only be used for transient transgenesis (Elroy-Stein, Fuerst et al. 1989; Elroy-Stein and Moss 1990). Thirdly, the vaccinia virus/bacteriophage RNAP hybrid expression system can be readily used in some cellular models that are permissive to vaccinia infection (e.g. BSC-1), whereas some are not (e.g. CHO). The insertion of the CP77 gene of cowpox virus into the genome of the recombinant vaccinia virus can overcome the vaccinia virus/bacteriophage RNAP hybrid expression system host range restriction of Chinese hamster ovary (CHO) cells by enabling the vaccinia virus to productively infect these cells (Spehner, Gillard et al. 1988; Ramsey-Ewing and Moss 1996). Fourthly, due to the complexity of the system, significant variability in its efficacy can be expected, even in the same cellular model. Fifthly, the vaccinia virus/bacteriophage RNAP hybrid expression system is a cost and labor-consuming technology, which is therefore poorly appropriate for large-scale assays and protein production.

In an attempt to couple capping to transcription and thus to improve the translatability of uncapped transcripts produced by the T7 RNA polymerase, this enzyme has been fused to the carboxyl-terminal domain (CTD) of the largest subunit of the RNA polymerase II (POLR2A), (Natalizio, Robson-Dixon et al. 2009). The CTD comprises 25-52 heptapeptide repeats of the consensus sequence ¹YSPTSPS⁷, which is highly conserved throughout evolution and subject to reversible phosphorylation during the transcription cycle (Palancade and Bensaude 2003). When phosphorylated, the CTD is thought to mediate the coupling of transcription and capping of nascent transcripts, by binding one or more subunits of the mRNA capping enzymes in yeast (Cho, Takagi et al. 1997; McCracken, Fong et al. 1997) and mammals (McCracken, Fong et al. 1997; Yue, Maldonado et al. 1997). Noticeably, RNA polymerase II with Ser⁵-phosphorylated CTD repeats undergoes promoter proximal pausing which is coincident with the co-transcriptional capping of the nascent transcripts (Komarnitsky, Cho et al. 2000; Schroeder, Schwer et al. 2000). However, in contrast to what could be expected intuitively, the fusion of the CTD to the single-unit T7 RNA polymerase is not sufficient to enhance the capping of both constitutively and alternatively spliced substrates in vivo (Natalizio, Robson-Dixon et al. 2009).

The capping is a specialized structure found at the 5′-end of nearly all eukaryotic messenger RNAs. The simplest cap structure, cap0, results of the addition of a guanine nucleoside methylated at N⁷ that is joined by 5′-5′ triphosphate bound to the end of primary RNA (i.e. m⁷GpppN where N is any base, p denotes a phosphate and m a methyl group). The formation of the cap0 involves a series of three enzymatic reactions: RNA triphosphatase (RTPase) removes the γ phosphate residue of 5′ triphosphate end of nascent pre-mRNA to diphosphate, RNA guanylyltransferase (GTase) transfers GMP from GTP to the diphosphate nascent RNA terminus, and RNA N7-guanine methyltransferase (N7-MTase) adds a methyl residue on azote 7 of guanine to the GpppN cap (Furuichi and Shatkin 2000). In higher eukaryotes and some viruses, the 2′-hydroxyl group of the ribose of the first (i.e. cap1 structures; m⁷GpppNm^(2′-O)pN) and second (i.e. cap2 structures; m⁷GpppNm^(2′-O)pNm^(2′-O) transcribed nucleotides can be methylated by two separate ribose-2′-O MTases, respectively named cap1- and cap2-specific MTases (Langberg and Moss 1981). However, In contrast to the cellular N7-MTase activity that is exclusively nuclear, cap1 ribose-2′-O MTase activity has been detected in both the cytoplasm and nucleus of HeLa cells, whereas cap2 MTase activity is exclusively found in their cytoplasm (Langberg and Moss 1981).

The formation of the 5′-terminal m⁷GpppN cap is the first step of pre-mRNA processing. The m⁷GpppN cap plays important roles in mRNA stability and its transport from the nucleus to the cytoplasm (Huang and Steitz 2005; Kohler and Hurt 2007). In addition, the 5′-terminal m⁷GpppN cap is important for the translation of mRNA to protein by anchoring the eukaryotic translation initiation factor 4F (eIF4F) complex, which mediates the recruitment of the 16S portion of the small ribosomal subunit to mRNA (Furuichi, LaFiandra et al. 1977; Gingras, Raught et al. 1999; Rhoads 1999). The 5′-terminal m⁷GpppN cap therefore enhances drastically the translation of mRNA both in vitro (Lo, Huang et al. 1998), and in vivo (Malone, Felgner et al. 1989; Gallie 1991; Lo, Huang et al. 1998; Kozak 2005). In contrast, the effects of 2′-O-methylation on mRNA translation appear to depend on the type of cells and the conditions of the experimentation (Epicentre Biotechnologies website; Drummond, Armstrong et al. 1985; Kuge, Brownlee et al. 1998).

There remains therefore a significant need in the art for new and improved systems for efficient transgenesis in eukaryotic cells, which are appropriate for gene therapy and large-scale protein production without cytotoxicity or induced-cytotoxicity. The present inventor has made a significant step forward with the invention disclosed herein.

The purpose of the invention is to fulfill this need by providing new chimeric enzymes, which make it possible to solve in whole or part the problems mentioned-above.

Unexpectedly, the inventor has demonstrated that chimeric enzymes comprising a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, a catalytic domain of a N⁷-guanine methyltransferase, and a catalytic domain of a DNA-dependant RNA polymerase are able to synthesize RNA molecules with 5′-terminal m⁷GpppN caps, which are highly translatable by the eukaryotic translational machinery, without cytotoxicity and while not inducing apoptosis.

These results are surprising since the capping of T7 transcripts remains infra-optimal with the vaccinia virus/bacteriophage RNAP hybrid expression system, and cannot be achieved by the fusion enzyme CTD-T7 RNA polymerase.

Thus, in one aspect, the invention relates to a chimeric enzyme comprising:

-   -   at least one, in particular a catalytic domain of a RNA         triphosphatase,     -   at least one, in particular a catalytic domain of a         guanylyltransferase,     -   at least one, in particular a catalytic domain of a N⁷-guanine         methyltransferase, and,     -   at least one, in particular a catalytic domain of a         DNA-dependant RNA polymerase.         In particular the chimeric enzyme according to the invention is         able to synthesize RNA molecules with 5′-terminal m⁷GpppN caps.

The chimeric enzyme according to the invention has in particular the following advantages:

-   -   There is no competition between the endogenous gene         transcription and the transgene transcription;     -   It is not expensive, quick and easy to implement and thus         appropriate for large-scale assays and protein production;     -   In contrast to the vaccinia virus/bacteriophage RNAP hybrid         expression system, it has no obvious cytotoxicity or         pro-apoptotic activities;     -   It allows the production of RNA transcripts in any eukaryotic         species (e.g. yeast, plants, rodents, dairy ruminants, primates,         and humans).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the relevant art.

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and claims are provided.

As used herein, the term “chimeric enzyme” refers to enzyme that is not a native enzyme that is found in nature. Accordingly, a chimeric enzyme may comprises catalytic domains that are derived from different sources (e.g. from different enzymes) or catalytic domains derived from the same source (e.g. from the same enzyme), but arranged in a different manner than that found in nature.

The term “chimeric enzyme” encompasses monomeric (i.e. single-unit) enzyme but also oligomeric (i.e. multi-unit) enzyme, in particular hetero-oligomeric enzyme.

As used herein, the term “monomeric enzyme” relates to a single-unit enzyme that consists of only one polypeptide chain.

As used herein, the term “oligomeric enzyme” refers to a multi-units enzyme that consists of at least two polypeptides chains, linked together covalently or noncovalently. The term “oligomeric enzyme” encompasses a multi-units enzyme, wherein at least two units of said enzyme are linked together covalently or noncovalently. The term “oligomeric enzyme” encompasses homo-oligomeric enzyme that is a multi-unit enzyme consisting of only one type of monomers (subunit) and hetero-oligomeric enzyme consisting of different types of monomers (subunits).

In particular, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase and of a DNA-dependant RNA polymerase are linked together covalently and/or noncovalently.

In particular, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase and of a DNA-dependant RNA polymerase are operatively linked together to synthesize RNA molecules with 5′-terminal m⁷GpppN caps.

In particular, the chimeric enzyme according to the invention comprising:

-   -   at least one, particularly a catalytic domain of a RNA         triphosphatase,     -   at least one, particularly a catalytic domain of a         guanylyltransferase,     -   at least one, particularly a catalytic domain of a N⁷-guanine         methyltransferase, and     -   at least one, particularly a catalytic domain of a DNA-dependant         RNA polymerase;

wherein at least two of said catalytic domains are linked together, preferably at their extremity (N or C terminal end), covalently or non-covalently, more particularly wherein at least one of the catalytic domain chosen in the group consisting of:

-   -   said at least one, particularly a catalytic domain of a RNA         triphosphatase,     -   said at least one, particularly a catalytic domain of a         guanylyltransferase, and     -   said at least one, particularly a catalytic domain of a         N⁷-guanine methyltransferase

is linked, preferably at its extremity (N or C terminal end), covalently or non-covalently with

-   -   said at least one, particularly a catalytic domain of a         DNA-dependant RNA polymerase, preferably at its extremity (N or         C terminal end).

In particular, the invention relates to the chimeric enzyme according to the invention comprising:

-   -   at least one, particularly a catalytic domain of a RNA         triphosphatase,     -   at least one, particularly a catalytic domain of a         guanylyltransferase,     -   at least one, particularly a catalytic domain of a N⁷-guanine         methyltransferase, and     -   at least one, particularly a catalytic domain of a DNA-dependant         RNA polymerase;     -   with the exception of chimeric enzyme comprising:     -   a catalytic domain of a RNA triphosphatase,     -   a catalytic domain of a guanylyltransferase,     -   a catalytic domain of a N⁷-guanine methyltransferase, and     -   only catalytic domains of nuclear eukaryotic DNA-dependant RNA         polymerase I, II and/or III; and more particularly, only         catalytic domain(s) of the DNA-dependant RNA polymerase II.

In particular, the invention relates to the chimeric enzyme according to the invention comprising:

-   -   at least one, particularly a catalytic domain of a RNA         triphosphatase,     -   at least one, particularly a catalytic domain of a         guanylyltransferase,     -   at least one, particularly a catalytic domain of a N⁷-guanine         methyltransferase, and     -   at least one, particularly a catalytic domain of a DNA-dependant         RNA polymerase;

with the exception of chimeric enzyme comprising:

-   -   a catalytic domain of a RNA triphosphatase,     -   a catalytic domain of a guanylyltransferase,     -   a catalytic domain of a N⁷-guanine methyltransferase, and     -   catalytic domains of nuclear eukaryotic DNA-dependant RNA         polymerase I, II and/or III, more particularly, at least a         catalytic domain of the DNA-dependant RNA polymerase II.

In particular, upon expression in a eukaryotic host cell, said chimeric enzyme according to the invention is able to synthesize RNA molecules with 5′-terminal m⁷GpppN cap, which are preferably translatable by the eukaryotic translational machinery.

Particularly, upon expression in a eukaryotic host cell said catalytic domains of a RNA triphosphatase, of a guanylyltransferase and of a N⁷-guanine methyltransferase are able to add a m⁷GpppN cap at the 5′-terminal end of RNA molecules synthesized by said catalytic domain of a DNA-dependant RNA polymerase and preferably said RNA molecules with 5′-terminal m⁷GpppN cap are translatable by the eukaryotic translational machinery.

Particularly, upon expression in a eukaryotic host cell, when said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a bacteriophage DNA-dependant RNA polymerase, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase and of a N⁷-guanine methyltransferase are able to add a m⁷GpppN cap at the 5′-terminal end of RNA molecules that have a guanosine ribonucleotide at their 5′ terminal end.

Particularly, upon expression in a eukaryotic host cell, when said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a bacterial DNA-dependant RNA polymerase, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase and of a N⁷-guanine methyltransferase are able to add a m⁷GpppN cap at the 5′-terminal end of RNA molecules that have a guanosine or a adenosine ribonucleotide at their 5′ terminal end.

Particularly, upon expression in a eukaryotic host cell, when said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a human or mouse mitochondrial DNA-dependant RNA polymerase, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase and of a N⁷-guanine methyltransferase are able to add a m⁷GpppN cap at the 5′-terminal end of RNA molecules that have a adenosine or a thymidine ribonucleotide at their 5′ terminal end.

A used herein the term “catalytic domain” of an enzyme relates to domain, which is necessary and sufficient, in particular in its three-dimensional structure, to assure the enzymatic function. For example, a catalytic domain of a RNA triphosphatase is the domain, which is necessary and sufficient to assure the RNA triphosphatase function. The term “catalytic domain” encompasses catalytic domain of wild type or mutant enzyme.

The chimeric enzyme according to the invention comprises at least said catalytic domains but can further comprise the whole or part of the enzymes containing said catalytic domains. In fact, according to one embodiment of the chimeric enzyme according to the invention, said catalytic domain of a DNA-dependant RNA polymerase can be included in the whole or part of a DNA-dependant RNA polymerase, preferably of a monomeric DNA-dependant RNA polymerase. Said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase and said catalytic domain of a N⁷-guanine methyltransferase can also be included in the whole or part of a capping enzyme, preferably of a monomeric capping enzyme.

The chimeric enzyme according to the invention can be a nuclear enzyme, a subcellular compartment enzyme or a cytoplasmic enzyme. Thus, the chimeric enzyme according to the invention can comprise a signal peptide or a marker-signal well known by one skilled in the art, which directs the transport of the enzyme in cells. For example, the chimeric enzyme according to the invention can comprise a nuclear localization signal (NLS), which directs the enzyme to the nucleus. Such NLS is often a unit consisting of five basic, plus-charged amino acids. The NLS can be located anywhere on the peptide chain.

Preferably, the chimeric enzyme according to the invention is a cytoplasmic chimeric enzyme. In particular, it does not comprise signal peptide or marker-signal that directs the transport of the enzyme, except to the cytoplasm.

The cytoplasmic localisation of the chimeric enzyme according to the invention has the advantage that it optimizes the levels of transgene expression by avoiding the active transfer of large DNA molecules (i.e. transgene) from the cytoplasm to the nucleus of eukaryotic cells and the export of RNA molecules from the nucleus to the cytoplasm.

These cytoplasmic chimeric enzymes according to the invention can thus be useful to generate a host-independent, eukaryotic gene expression system that is able to work in the cytoplasm in which significantly higher amounts of transfected DNA are usually found as compared to the nucleus.

These cytoplasmic chimeric enzymes according to the invention are able to synthesize RNA molecules with 5′-terminal m⁷GpppN caps, which are highly translatable by the eukaryotic cytoplasmic translational machinery, without cytotoxicity and while not inducing apoptosis.

There is also no competition between the endogenous gene transcription and the transgene transcription, since the endogenous gene transcription occurs in the nucleus of eukaryotic cells in contrast to the transgene transcription, which occurs in the cytoplasm.

The cytoplasmic chimeric enzyme according to the invention is thus notably appropriate for large-scale assays and protein production.

In one embodiment of the chimeric enzyme according to the invention, said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase, said catalytic domain of a N⁷-guanine methyltransferase, are included in a monomer, i.e. in one polypeptide. For example, said monomer can be a monomeric capping enzyme or a monomeric chimeric enzyme according to the invention.

In particular, said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase, and said catalytic domain of a N⁷-guanine methyltransferase are included in a monomeric capping enzyme. In this case, the chimeric enzyme according to the invention comprise a monomeric capping enzyme, which includes said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase, and said catalytic domain of a N⁷-guanine methyltransferase. Said monomeric capping enzyme can be a monomeric virus capping enzyme, in particular chosen in the group consisting of the wild type bluetongue virus capping enzyme, the wild type bamboo mosaic virus capping enzyme, the wild type African swine fever virus capping enzyme, the wild type acanthamoeba polyphaga mimivirus capping enzyme and mutants and derivatives thereof which are able to add a m⁷GpppN cap at the 5′-terminal end of RNA molecules and, more particularly of the wild type African swine fever virus capping enzyme and mutants and derivatives thereof which are able to add a m⁷GpppN cap at the 5′-terminal end of RNA molecules, and even more particularly the wild type African swine fever virus capping enzyme.

In particular, said catalytic domain of a DNA-dependant RNA polymerase can also be included in a monomer, i.e. in one polypeptide. For example, said monomer can be a monomeric DNA-dependent RNA polymerase or a monomeric chimeric enzyme according to the invention.

In particular, said catalytic domain of a DNA-dependant RNA polymerase is included in a monomeric DNA-dependent RNA polymerase. In this case, the chimeric enzyme according to the invention comprises a monomeric DNA-dependent RNA polymerase, which includes said catalytic domain of a DNA-dependant RNA polymerase. Said monomeric DNA-dependent RNA polymerase can be a monomeric phage DNA-dependent RNA polymerase, in particular chosen in the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase and mutants or derivatives thereof, which are able to synthesize single-stranded RNA complementary in sequence to the double-stranded template DNA in the 5′→3′ direction, more particularly of T7 RNA polymerase and mutants or derivatives thereof, which are able to synthesize single-stranded RNA complementary in sequence to the double-stranded template DNA in the 5′→3′ direction.

Said catalytic domain of a DNA-dependant RNA polymerase and at least one, preferably at least two and more preferably the whole catalytic domain chosen in the group consisting of:

-   -   said catalytic domain of a RNA triphosphatase;     -   said catalytic domain of a guanylyltransferase; and     -   said catalytic domain of a N⁷-guanine methyltransferase;

can be included in a monomer.

The chimeric enzyme according to the invention can be monomeric or oligomeric. In fact, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase and of a DNA-dependant RNA polymerase can be included in one or several polypeptides.

Preferably, the chimeric enzyme according to the invention is monomeric.

In fact, the inventor has demonstrated that a monomeric chimeric enzyme comprising a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, a catalytic domain of a N⁷-guanine methyltransferase, and a catalytic domain of a DNA-dependant RNA polymerase are able to synthesize RNA molecules with 5′-terminal m⁷GpppN caps, which are highly translatable by the eukaryotic translational machinery, without cytotoxicity and while not inducing apoptosis.

It was not obvious that the capping of transcripts well occurred with a monomeric enzyme, due to steric hindrance and components and the enzyme, which have to remain in their native conformation. In fact, the capping of T7 transcripts cannot be achieved by the fusion enzyme CTD-T7 RNA polymerase, although it was supposed to trigger m⁷GpppN capping at the 5′-terminal end of nascent RNA molecules.

The monomeric chimeric enzyme according to the invention has in particular the advantages that it is not expensive, quick and easy to implement and thus appropriate notably for large-scale assays and protein production. In fact, the production of a monomeric enzyme is easier than of oligomeric enzyme. There is also no problem of unit assembly, since there is only a single-unit. The monomeric enzyme is also easier to manipulate than multimeric enzyme.

As used herein, the term “DNA-dependent RNA polymerase” (RNAPs) relates to nucleotidyl transferases that synthesize single-stranded RNA complementary in sequence to the double-stranded template DNA in the 5′→3′ direction.

Preferably, said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of an enzyme, which have a relatively simple structure and more preferably, which have characterized genomic enzymatic regulation elements (i.e. promoter, transcription termination signal and concatemer junction). Thus, in particular, said catalytic domain of a DNA-dependant RNA polymerase can be a catalytic domain of a bacteriophage DNA-dependant RNA polymerase, of a bacterial DNA-dependant RNA polymerase or of a DNA-dependant RNA polymerase of various eukaryotic organelles (e.g. mitochondria, chloroplast and proplastids).

In one embodiment, said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a bacteriophage DNA-dependant RNA polymerase.

The bacteriophage DNA-dependant RNA polymerases have notably the advantage that they optimize the levels of transgene expression, in particular by having a higher processivity than the eukaryotic RNA polymerases. The bacteriophage DNA-dependant RNA polymerases have also a much simpler structure than most nuclear eukaryotic polymerases, which have complex structure with multiple subunits (e.g. RNA polymerase II) (Chen and Schneider 2005). Most of the bacteriophage DNA-dependant RNA polymerases characterized so far are single-subunit enzymes, which require no accessory proteins for initiation, elongation, or termination of transcription (Chen and Schneider 2005). Several of these enzymes, which are named for the bacteriophages from which they have been cloned, have also well-characterized regulation genomic elements (i.e. promoter, termination signals, transcriptional pausing sequences), which are important for transgenesis.

There is also no competition between the endogenous gene transcription and the transgene transcription. The chimeric enzymes according to the invention, which comprise bacteriophage DNA dependant RNA-polymerase moieties allow the production of RNA transcripts in any eukaryotic species (e.g. yeast, rodents, and humans). They are not expensive, quick and easy to implement and thus appropriate for large-scale assays and protein productions; it allows the production of RNA transcripts in any biological system (e.g. acellular reaction mix, cultured cells, and living organisms), since in contrast to eukaryotic RNA polymerase such as RNA polymerase II, most of bacteriophage DNA dependant RNA polymerases do not require associated factors for initiation, elongation or termination of transcription.

Said catalytic domain of a bacteriophage DNA-dependant RNA polymerase can be a catalytic domain of a bacteriophage DNA-dependant RNA polymerase, in particular chosen in the group consisting of the wild type of the T7 RNA polymerase, the wild type of the T3 RNA polymerase (NCBI genomic sequence ID# NC_003298; GeneID#927437; UniProtKB/Swiss-Prot ID# Q778M8), the wild type of the K11 RNA polymerase (NCBI genomic K11 RNAP sequence ID# NC_004665; GeneID#1258850; UniProtKB/Swiss-Prot ID# Q859H5), the wild type of the φA1122 RNA polymerase (NCBI genomic sequence ID# NC_004777; GeneID#1733944; UniProtKB/Swiss-Prot protein ID# Q858N4), the wild type of the φYeo3-12 RNA polymerase (NCBI genomic sequence ID# NC_001271; GeneID#1262422; UniProtKB/Swiss-Prot ID# Q9T145) and the wild type of the gh-1 RNA polymerase (NCBI genomic sequence ID# NC_004665; GeneID#1258850; UniProtKB/Swiss-Prot protein ID# Q859H5), the wild type of the K1-5 RNAP RNA polymerase (NCBI genomic sequence ID# NC_008152; GeneID#5075932; UniProtKB/Swiss-Prot protein ID# Q8SCG8) and the wild type of the SP6 RNA polymerase (NCBI genomic sequence ID# NC_004831; GeneID#1481778; UniProtKB/Swiss-Prot protein ID# Q7Y5R1), and mutants or derivatives thereof, which are able to synthesize single-stranded RNA complementary in sequence to the double-stranded template DNA in the 5′→3′ direction, more particularly of the wild type of T7 RNA polymerase.

As used herein, the term “T7 RNA polymerase” relates to the bacteriophage T7 DNA-dependant RNA polymerase. Preferably, the T7 RNA polymerase have the amino acid sequence of SEQ ID NO:1 (NCBI genomic sequence ID# NC_001604; GeneID#1261050; UniProtKB/Swiss-Prot ID# P00573) and is a 883 amino-acid protein with a molecular weight of 98.8 kDa (Davanloo, Rosenberg et al. 1984; Moffatt, Dunn et al. 1984).

The T7 RNA polymerase has in particular the advantage that, in vitro, the enzyme is extremely processive and elongates 240-250 nucleotides/s at 37° C. in the 5′→3′ direction (Golomb and Chamberlin 1974; Lyakhov, He et al. 1997; Zhang and Studier 1997; Finn, MacLachlan et al. 2005). Moreover, when expressed in eukaryotic cells, the T7 RNA polymerase, remains largely in the cytoplasm (Elroy-Stein and Moss 1990; Gao and Huang 1993; Brisson, He et al. 1999), and thus optimizes the levels of transgene expression by avoiding the active transfer of large DNA molecules (i.e. transgene) from the cytoplasm to the nucleus of eukaryotic cells and the export of RNA molecules from the nucleus to the cytoplasm.

The catalytic domain of a DNA-dependant RNA polymerase can be the one of the wild-type of the T7 RNA polymerase but also of mutants of the T7 RNA polymerase, which are able to synthesize single-stranded RNA complementary in sequence to the double-stranded template DNA in the 5′→3′ direction, even with reduced processivity. For example, said mutants can be chosen in the group comprising R551S, F644A, Q649S, G645A, R627S, I810S, and D812E (Makarova, Makarov et al. 1995), and K631M (Osumi-Davis, de Aguilera et al. 1992; Osumi-Davis, Sreerama et al. 1994).

In one embodiment of the chimeric enzyme according to the invention, said catalytic domain of a DNA-dependant RNA polymerase is located C-terminally with respect to said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase, and said catalytic domain of a N⁷-guanine methyltransferase.

In fact, when the catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a bacteriophage DNA-dependant RNA polymerase, in particular of a bacteriophage DNA-dependant RNA polymerase chosen in the group consisting of T7, T3 and SP6-RNA polymerases, said catalytic domain preferably conserves its native carboxyl-terminal end. In particular, the C-terminal end of said catalytic domain of a DNA-dependant RNA polymerase, particularly of a bacteriophage DNA-dependant RNA polymerase corresponds to the C-terminal end of said chimeric enzyme. In particular, when the chimeric enzyme comprises a whole bacteriophage DNA-dependant RNA polymerase, in particular of a bacteriophage DNA-dependant RNA polymerase chosen in the group consisting of T7, T3 and SP6-RNA polymerases, said polymerase preferably conserves its native carboxyl-terminal end. In particular, the C-terminal end of said catalytic domain of a DNA-dependant RNA polymerase, particularly of a bacteriophage DNA-dependant RNA polymerase corresponds to the C-terminal end of said chimeric enzyme. Particularly, said catalytic domain of a DNA-dependant RNA polymerase, in particular of a bacteriophage DNA-dependant RNA polymerase chosen in the group consisting of T7, T3 and SP6-RNA polymerases, is included in the whole or part of a bacteriophage DNA-dependant RNA polymerase and wherein the C-terminal end of said bacteriophage DNA-dependant RNA polymerase corresponds to the C-terminal end of said chimeric enzyme.

In one embodiment of the chimeric enzyme according to the invention, said catalytic domain of a DNA-dependant RNA polymerase, particularly said catalytic domain of a bacteriophage DNA-dependant RNA polymerase, in particular of a bacteriophage DNA-dependant RNA polymerase chosen in the group consisting of T7, T3 and SP6-RNA polymerases, is located C-terminally with respect to said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase, and said catalytic domain of a N⁷-guanine methyltransferase.

In another embodiment, said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a bacterial DNA-dependant RNA polymerase.

Preferably said bacterial DNA-dependant RNA polymerase has a moderate structure complexity.

For example, said bacterial DNA-dependant RNA polymerase can be the E. coli DNA-dependent RNA polymerase (NCBI genomic sequence of K-12 substrain DH10B ID# NC_010473), which consists of four different subunits (a subunit: rpoA GeneID#6060938, UniProtKB/Swiss-Prot ID# B1X6E7; β subunit: rpoB GeneID#6058462, UniProtKB/Swiss-Prot ID# B1XBY9; β′ subunit: rpoC GeneID#6058956, UniProtKB/Swiss-Prot ID# B1XBZ0; σ subunit: rpoE GeneID#6060683, UniProtKB/Swiss-Prot ID# B1XBQ0), which are assembled in a five ααββ′σ subunit complex (Lodish, Berk et al. 2008). The genomic elements involved in the regulation of the enzymatic activity are well-characterized, including E. coli RNA polymerase promoters (Lisser and Margalit 1993), termination signals including rho-dependant and -independent terminators (Platt 1986; Uptain and Chamberlin 1997), and transcriptional pausing sequences (Lee, Phung et al. 1990).

In another embodiment, said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a DNA-dependant RNA polymerase of a eukaryotic organelle, like mitochondria, chloroplast and proplastids. In fact, these polymerases can also have relatively simple structure.

In particular, said catalytic domain of a DNA-dependant RNA polymerase is a catalytic domain of a mitochondrial DNA-dependant RNA polymerase.

Particularly, said catalytic domain of a DNA-dependant RNA polymerase can be the catalytic domain of the mammalian mouse mitochondrial RNA polymerase, which is a single-unit 120 kDa protein (GeneID#216151, UniProtKB/Swiss-Prot ID# Q8BKF1), which shares homology with the bacteriophage RNA polymerases (Tiranti, Savoia et al. 1997). Several transcription factors are required for transcription initiation, elongation, or termination: TFB1M (mitochondrial transcription factor B1; mouse GeneID#224481, UniProtKB/Swiss-Prot ID# Q8JZM0) or TFB2M (mitochondrial transcription factor B2; mouse GeneID#15278, UniProtKB/Swiss-Prot ID# Q3TL26), TFAM (mitochondrial transcription factor A; mouse GeneID#21780, UniProtKB/Swiss-Prot ID# P40630), and mTERF (mitochondrial transcription termination factor; mouse GeneID#545725, UniProtKB/Swiss-Prot ID# Q8CHZ9) for termination of transcription (Fisher and Clayton 1985; Fisher, Topper et al. 1987; Fisher and Clayton 1988; Topper and Clayton 1989; Fernandez-Silva, Martinez-Azorin et al. 1997; Prieto-Martin, Montoya et al. 2001; McCulloch, Seidel-Rogol et al. 2002). The genomic elements involved in the regulation of the enzymatic activity of the mitochondrial RNA polymerase are well-characterized, including two promoters at light- and heavy-strands of the mitochondrial genome (Ojala, Montoya et al. 1981; Clayton 1991), as well as transcriptional termination signals (Kruse, Narasimhan et al. 1989).

As used herein, the term “RNA triphosphatase” (RTPase) relates to the enzyme, which removes the γ phosphate residue of 5′ triphosphate end of nascent pre-mRNA to diphosphate (Furuichi and Shatkin 2000).

As used herein, the term “RNA guanylyltransferase” (GTase) refers to the enzyme, which transfers GMP from GTP to the diphosphate nascent RNA terminus (Furuichi and Shatkin 2000).

As used herein, the term “N7-guanine methyltransferase” (N7-MTase) relates to the enzyme, which adds a methyl residue on azote 7 of guanine to the GpppN cap (Furuichi and Shatkin 2000).

Said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase, can be of the same or of different capping enzymes. If said catalytic domains are of the same enzyme, said catalytic domain of a DNA-dependant RNA polymerase is of a different enzyme.

Preferably, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase are from one or several cytoplasmic enzymes, which have advantageously relatively simple structure and well-characterized enzymatic activities. Thus, in particular, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase can be catalytic domains of one or several virus capping enzymes, or of capping enzymes of cytoplasmic episomes.

In one embodiment, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase are from one or several virus capping enzymes, in particular chosen in the group consisting of the wild type bluetongue virus capping enzyme, the wild type bamboo mosaic virus capping enzyme, the wild type African swine fever virus capping enzyme, the wild type acanthamoeba polyphaga mimivirus capping enzyme, and mutants or derivatives thereof which are able respectively to remove the γ phosphate residue of 5′ triphosphate end of nascent pre-mRNA to diphosphate or transfer GMP from GTP to the diphosphate nascent RNA terminus or add a methyl residue on azote 7 of guanine to the GpppN cap, more particularly of the wild type African swine fever virus capping enzyme.

As used herein the term “bluetongue virus capping enzyme” relates to the single-unit VP4 capping enzyme of Bluetongue virus (BTV), which is a 76 kDa protein (644 amino-acids; for sequence, see for instance NCBI BTV serotype 10 genomic sequence ID# Y00421; GeneID#2943157; UniProtKB/Swiss-Prot ID# P07132, D0UZ45, Q5J7C0, Q65751, Q8BA65, P33428, P33429, P33427, C3TUP7, Q8BAD5, C5IWW0, B4E551, Q3KVQ2, Q3KVQ1, Q65732, Q3KVP8, Q3KVP9, Q3KVQ0). This capping enzyme is likely able to homodimerize through the leucine zipper located near its carboxyl-terminus (Ramadevi, Rodriguez et al. 1998). VP4 catalyze all three enzymatic steps required for mRNA m⁷GpppN capping synthesis: RTPase (Martinez-Costas, Sutton et al. 1998), GTase (Martinez-Costas, Sutton et al. 1998; Ramadevi, Burroughs et al. 1998) and N7-MTase (Ramadevi, Burroughs et al. 1998).

As used herein, the term “bamboo mosaic virus capping enzyme” relates to ORF1, the Bamboo Mosaic Virus (BMV) mRNA capping enzyme, which is a single-unit 155-kDa protein (1365-amino acids; NCBI BMV isolate BaMV-O genomic sequence ID# NC_001642; GeneID#1497253; UniProtKB/Swiss-Prot ID# Q65005). ORF1 protein has all the enzymatic activities required to generate m⁷GpppN mRNA capping, i.e. RTPase (Li, Shih et al. 2001; Han, Tsai et al. 2007), GTase and N7-MTase (Li, Chen et al. 2001; Li, Shih et al. 2001). In addition, ORF1 has RNA-dependent RNA-polymerase activity, which is not mandatory for chimeric enzymatic activities according to the invention and can be abolished by deletion of Asp¹²²⁹Asp¹²³⁰ residues of the mRNA capping enzyme (Li, Cheng et al. 1998). As used herein, the term “African swine fever virus capping enzyme” relates to the NP868R capping enzyme (G4R), (ASFV), which is a single-unit 100 kDa protein (868 amino-acids; NCBI ASFV genomic sequence strain BA71V ID# NC_001659; GeneID#1488865; UniProtKB/Swiss-Prot ID# P32094).

As used herein, the term “acanthamoeba polyphaga mimivirus capping enzyme” relates to R382, (APMV), which is another single-unit 136.5 kDa protein (1170 amino-acids; NCBI APMV genomic sequence ID# NC_006450; GeneID#3162607; UniProtKB/Swiss-Prot ID# Q5UQX1).

In one embodiment, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase are from one or several capping enzymes of cytoplasmic episomes, like pGKL2. In particular, said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase are included in the whole or part of the capping enzyme of the yeast linear extra-chromosomal episome pGKL2.

Cytoplasmic linear episomes are double-stranded DNA structures, which stably replicate in the cytoplasm of various yeast strains (Cong, Yarrow et al. 1994). One prototype of yeast linear extra-chromosomal episome, pGKL2 (13,457 bp; also named pGK12), has been entirely sequenced from various yeast strains, including Kluyveromyces lactis CB 2359 and Saccharomyces cerevisiae F102-2 (Tommasino, Ricci et al. 1988). The capping enzyme encoded by the ORF3 gene of Kluyveromyces lactis pGKL2 (NCBI Kluyveromyces lactis CB 2359 pGKL2 genomic sequence ID# NC_010187; UniProtKB/Swiss-Prot ID# P05469) is a 594 amino-acid protein (70.6 kDa protein).

In one embodiment of the chimeric enzyme according to the invention, at least two, in particularly at least three and more particularly the whole catalytic domains can be assembled, fused, or bound directly or indirectly by a linking peptide.

In particular at least two, particularly at least three and more particularly the whole catalytic domains chosen in the group consisting of:

-   -   a catalytic domain of a RNA triphosphatase,     -   a catalytic domain of a guanylyltransferase,     -   a catalytic domain of a N⁷-guanine methyltransferase, and     -   a catalytic domain of a DNA-dependant RNA polymerase.

are bound directly or by a linking peptide.

Linking peptide has the advantage of generating fusion proteins in which steric hindrance is minimizes and enough space is provided for the components of the fusion protein to remain in their native conformation.

Preferably, at least said catalytic domain of a DNA-dependant RNA polymerase is bound by a linking peptide to at least one of the catalytic domain chosen in the group consisting of:

-   -   said catalytic domain of a RNA triphosphatase;     -   said catalytic domain of a guanylyltransferase; and     -   said catalytic domain of a N⁷-guanine methyltransferase.

Particularly, the linking peptide can be located N-terminally with respect to said catalytic domain of a DNA-dependant RNA polymerase, in particular of a bacteriophage DNA-dependant RNA polymerase chosen in the group consisting of T7, T3 and SP6-RNA polymerases, and C-terminally with respect to said catalytic domain of a RNA triphosphatase, said catalytic domain of a guanylyltransferase, and said catalytic domain of a N⁷-guanine methyltransferase.

In particular, the N-terminal end of said catalytic domain of a DNA-dependant RNA polymerase, in particular of a bacteriophage DNA-dependant RNA polymerase chosen in the group consisting of T7, T3 and SP6-RNA polymerases, is linked by covalent linkage, in particular by a linking peptide, to the C-terminal end of one of the catalytic domain chosen in the group consisting of:

-   -   said catalytic domain of a RNA triphosphatase,     -   said catalytic domain of a guanylyltransferase, and     -   said catalytic domain of a N⁷-guanine methyltransferase.

Said linking peptide can be chosen from the group consisting of:

-   -   peptides of formula (Gly_(m)Ser_(p))_(n), in which:         -   m represents an integer from 0 to 12, in particular from 1             to 8, and more particularly from 3 to 6 and even more             particularly 4;         -   p represents an integer from 0 to 6, in particular from 0 to             5, more particularly from 0 to 3 and more particularly 1;             and         -   n represents an integer from 0 to 30, in particular from 0             to 12, more particularly from 0 to 8 and even more             particularly between 1 and 6 inclusive;     -   peptides consisting of an amino acid sequence selected from the         group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ         ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ         ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID         NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

The flexible linker peptides of formula (Gly_(m)Ser_(p))_(n) have the advantages that the glycine residues confer peptide flexibility, while the serine provide some solubility (Huston, Levinson et al. 1988). Furthermore, the absence of sensitive sites for chymotrypsin I, factor Xa, papain, plasmin, thrombin and trypsin in the (Gly_(m)Ser_(p))_(n) linker sequences is supposed to increase the overall stability of the resulting fusion proteins (Crasto and Feng 2000).

(Gly_(m)Ser_(p))_(n) linkers of variable lengths are commonly used to engineer single-chain Fv fragment (sFv) antibodies (Huston, Levinson et al. 1988). In addition, (Gly_(m)Ser_(p))_(n) linkers have been used to generate various fusion proteins, which frequently retain the biological activities of each of their components (Newton, Xue et al. 1996; Lieschke, Rao et al. 1997; Shao, Zhang et al. 2000; Hu, Li et al. 2004).

Other types of peptide linkers can be also considered to generate chimeric enzymes according to the invention, such as GGGGIAPSMVGGGGS (SEQ ID NO:2) (Turner, Ritter et al. 1997), SPNGASNSGSAPDTSSAPGSQ (SEQ ID NO:3) (Hennecke, Krebber et al. 1998), EGKSSGSGSESKSTE (SEQ ID NO:4) (Bird, Hardman et al. 1988), EGKSSGSGSESKEF (SEQ ID NO:5) (Newton, Xue et al. 1996), GGGSGGGSGGGTGGGSGGG (SEQ ID NO:6) (Robinson and Sauer 1998), GSTSGSGKSSEGKG (SEQ ID NO:7) (Bedzyk, Weidner et al. 1990), YPRSIYIRRRHPSPSLTT (SEQ ID NO:8) (Tang, Jiang et al. 1996), GSTSGSGKPGSGEGSTKG (SEQ ID NO:9) (Whitlow, Bell et al. 1993), GSTSGSGKPGSGEGS (SEQ ID NO:10) (Ting, Kain et al. 2001), SSADDAKKDAAKKDDAKKDDAKKDA (SEQ ID NO:11) (Pantoliano, Bird et al. 1991), GSADDAXXDAAXKDDAKKDDAKKDGS (SEQ ID NO:12) (Gregoire, Lin et al. 1996), LSADDAKKDAAKKDDAKKDDAKKDL (SEQ ID NO:13) (Pavlinkova, Beresford et al. 1999), AEAAAKEAAAKEAAAKA (SEQ ID NO:14) (Wickham, Carrion et al. 1995), GSHSGSGKP (SEQ ID NO:15) (Ting, Kain et al. 2001), GSTSGSGKPGSGEGSTGAGGAGSTSGSGKPSGEG (SEQ ID NO:16) (Ting 2003), LSLEVAEEIARLEAEV (SEQ ID NO:17) (Liu, Jian-Bo et al. 2005), and GTPTPTPTPTGEF (SEQ ID NO:18) (Gustavsson, Lehtio et al. 2001).

Other types of covalent linkage include but are not limited to disulfide bounds (Mantile, Fuchs et al. 2000), transglutamination (Paguirigan and Beebe 2007), as well as protein trans-linking by chemical and/or physical agents, e.g. cross-linking by tris(bipyridine)ruthenium(II)-dichloride and ultraviolet light illumination (Fancy and Kodadek 1999).

Said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase, and of a DNA-dependant RNA polymerase can also be assembled by specific protein elements, like leucine zippers, like biotinylation domain to one of the catalytic domain (e.g. Avi-tag II (Cronan 1990) or PFB-tag (Wu, Yeung et al. 2002)) and a biotin binding domain to one of the other catalytic domain (e.g. Strep-tag II (Schmidt and Skerra 1993) or Nano-tag (Lamla and Erdmann 2004)) in the chimeric enzyme according to the invention.

In one embodiment of the chimeric enzyme according to the invention, at least two of said catalytic domains can be assembled, by non-covalent linkage, in particular by leucine zippers.

Preferably, at least said catalytic domain of a DNA-dependant RNA polymerase is assembled by non-covalent linkage, in particular by leucine zippers, to at least one of the catalytic domain chosen in the group consisting of:

-   -   said catalytic domain of a RNA triphosphatase;     -   said catalytic domain of a guanylyltransferase; and     -   said catalytic domain of a N⁷-guanine methyltransferase.

The leucine zippers, which are dimeric coiled-coil protein structures composed of two amphipathic α-helices that interact with each other, are commonly used to homo- or hetero-dimerize proteins (O'Shea, Klemm et al. 1991). Each helices consist of repeats of seven amino acids, in which the first amino-acid (residue a) is hydrophobic, the fourth (residue d) is usually a Leucine, while the other residues are polar. The leucine zippers VELCRO ACID-p1 and BASE-p1, which form a parallel heterodimeric two-stranded coiled coil structures, have high propensity to form parallel protein hetero-dimers (O'Shea, Lumb et al. 1993). They have been used to heterodimerize membrane proteins (Chang, Bao et al. 1994; Pashine, Busch et al. 2003), as well as several soluble proteins (Busch, Reich et al. 1998; Busch, Pashine et al. 2002).

Other types of oligomerisation peptide domains can be also considered to generate chimeric enzyme according to the invention, to assemble at least two of said catalytic domains of the chimeric enzyme according to the invention, especially leucine zippers that form antiparallel heteromeric structures, such as the ACID-a1/BASE-a1 (Oakley and Kim 1998), ACID-Kg/BASE-Eg (McClain, Woods et al. 2001), NZ/CZ (Ghosh, Hamilton et al. 2000), ACID-pLL/BASE-pLL (Lumb and Kim 1995), and EE₁₂₃₄L and RR₁₂₃₄L (Moll, Ruvinov et al. 2001) leucine zippers. Disulfide-linked versions of leucine zippers can be also used to generate disulfide coiled coil-bound heterodimeric chimeric enzyme according to the invention (O'Shea, Lumb et al. 1993), as well as interchain disulfide bridges between cysteine residues under oxidizing conditions (Wells and Powers 1986).

At least two of said catalytic domains of a RNA triphosphatase, of a guanylyltransferase, of a N⁷-guanine methyltransferase, and of a DNA-dependant RNA polymerase can thus be assembled by leucine zippers, in particular leucine zippers that form antiparallel heteromeric structures, such as the ACID-a1/BASE-a1 (Oakley and Kim 1998), ACID-Kg/BASE-Eg (McClain, Woods et al. 2001), NZ/CZ (Ghosh, Hamilton et al. 2000), and ACID-pLL/BASE-pLL leucine zippers, disulfide coiled coil-bound (O'Shea, Lumb et al. 1993), as well as disulfide bridges between cysteine residues (Wells and Powers 1986).

In one embodiment, the chimeric enzyme according to the invention comprises:

-   -   the wild type mRNA capping enzyme of the African Swine Fever         virus or a mutant or a derivative thereof, which is able to add         a m⁷GpppN cap at the 5′-terminal end of RNA molecules, in         particular the wild type African swine fever virus capping         enzyme, fused to     -   the amino-terminal end of the wild type T7 RNA polymerase or         mutant or derivative thereof which is able to synthesize         single-stranded RNA complementary in sequence to the         double-stranded template DNA in the 5′→3′ direction, in         particular the wild type of T7 RNA polymerase,

in particular via a linker, and more particularly via a (Gly₃Ser)₄ linker.

In another embodiment, the chimeric enzyme according to the invention comprises:

-   -   the wild type mRNA capping enzyme of the African Swine Fever         virus or a mutant or a derivative thereof, which is able to add         a m⁷GpppN cap at the 5′-terminal end of RNA molecules, in         particular the wild type African swine fever virus capping         enzyme, fused to     -   the amino-terminal end of the wild type T3 RNA polymerase or         mutant or derivative thereof which is able to synthetize         single-stranded RNA complementary in sequence to the         double-stranded template DNA in the 5′→3′ direction, in         particular the wild type of T3 RNA polymerase, in particular via         a linker, and more particularly via a (Gly₃Ser)₄ linker.

In another embodiment, the chimeric enzyme according to the invention comprises:

-   -   the wild type mRNA capping enzyme of the African Swine Fever         virus or a mutant or a derivative thereof, which is able to add         a m⁷GpppN cap at the 5′-terminal end of RNA molecules, in         particular the wild type African swine fever virus capping         enzyme, fused to     -   the amino-terminal end of the wild type SP6 RNA polymerase or         mutant or derivative thereof which is able to synthetize         single-stranded RNA complementary in sequence to the         double-stranded template DNA in the 5′→3′ direction, in         particular the wild type of SP6 RNA polymerase,

in particular via a linker, and more particularly via a (Gly₃Ser)₄ linker.

In another embodiment, the chimeric enzyme according to the invention comprises

-   -   the wild type mRNA capping enzyme of the African Swine Fever         virus or a mutant or a derivative thereof, which is able to add         a m⁷GpppN cap at the 5′-terminal end of RNA molecules, in         particular the wild type African swine fever virus capping         enzyme, and     -   the amino-terminal end of the wild type T7 RNA polymerase or         mutant or derivative thereof which is able to synthetize         single-stranded RNA complementary in sequence to the         double-stranded template DNA in the 5′→3′ direction, in         particular the wild type of T7 RNA polymerase,

assembled by leucine zippers.

The chimeric enzyme according to the invention can also further comprise a domain, which enhance the activity of at least one catalytic domain of the chimeric enzyme of the invention, in particular of at least one catalytic domain chosen in the group consisting of a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, a catalytic domain of a N⁷-guanine methyltransferase and a catalytic domain of a DNA-dependant RNA polymerase.

For example said domain, which enhance the activity of at least one catalytic domain of the chimeric enzyme of the invention, can be a 31-kDa subunit encoded by the vaccinia virus D12L gene (genomic sequence ID# NC_006998.1; GeneID#3707515; UniProtKB/Swiss-Prot ID#YP_232999.1), which has no intrinsic enzymatic activity, but enhances drastically the RNA N7-guanine methyltransferase activity of the D1R subunit of the vaccinia mRNA capping enzyme (Higman, Bourgeois et al. 1992; Higman, Christen et al. 1994; Mao and Shuman 1994).

In one embodiment, the chimeric enzyme of the invention comprises:

-   -   at least one catalytic domain of the vaccinia mRNA capping         enzyme, in particular the 95 kDa subunit encoded by the vaccinia         virus D1R gene (genomic sequence ID# NC_006998.1;         GeneID#3707562; UniProtKB/Swiss-Prot ID# YP_232988.1), which has         RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine         methyltransferase enzymatic activities (Cong and Shuman 1993;         Niles and Christen 1993; Higman and Niles 1994; Mao and Shuman         1994; Gong and Shuman 2003);     -   at least one catalytic domain of a DNA-dependant RNA polymerase,         in particular chosen in the group consisting of T7, T3 and         SP6-RNA polymerases and more particularly the T7 RNA polymerase;         and     -   a 31-kDa subunit encoded by the vaccinia virus D12L gene,

in particular assembled in whole or part via a linker, and more particularly via a (Gly₃Ser)₄ linker and/or by leucine zippers.

The invention also relates to an isolated nucleic acid molecule or a group of isolated nucleic acid molecules, said nucleic acid molecule(s) encoding a chimeric enzyme according to the invention.

Said group of isolated nucleic molecules encoding a chimeric enzyme according to the invention comprises or consists of all the nucleic acid molecules which are necessary and sufficient to obtain a chimeric enzyme according to the invention by their expression.

In one embodiment, said group of isolated nucleic acid molecules encoding a chimeric enzyme according to the invention comprises or consists of:

-   -   a nucleic acid molecule encoding at least one catalytic domain         of a RNA triphosphatase, at least one catalytic domain of a         guanylyltransferase and at least one catalytic domain of a         N⁷-guanine methyltransferase; and     -   a nucleic acid molecule encoding at least one catalytic domain         of a DNA-dependant RNA polymerase.

In another embodiment, said group of isolated nucleic acid molecules encoding a chimeric enzyme according to the invention comprises or consists of:

-   -   a nucleic acid molecule encoding at least one catalytic domain         of a RNA triphosphatase,     -   a nucleic acid molecule encoding at least one catalytic domain         of a guanylyltransferase,     -   a nucleic acid molecule encoding at least one catalytic domain         of a N⁷-guanine methyltransferase; and     -   a nucleic acid molecule encoding at least one catalytic domain         of a DNA-dependant RNA polymerase.

In particular, the nucleic acid molecule according to the invention can be operatively linked to at least one, preferably the whole promoter(s) chosen from the group consisting of:

-   -   a promoter for an eukaryotic DNA dependant RNA polymerase,         preferably for RNA polymerase II; and     -   a promoter for said catalytic domain of a DNA-dependant RNA         polymerase.

The link of the nucleic acid to a promoter for a eukaryotic DNA dependant RNA polymerase, preferably for RNA polymerase II has notably the advantage that when the chimeric enzyme of the invention is expressed in an eukaryotic host cell, the expression of the chimeric enzymes is driven by the eukaryotic RNA polymerase, preferably the RNA polymerase II. These chimeric enzymes, in turn, can initiate transcription of the transgene. If tissue-specific RNA polymerase II promoters are used, the chimeric enzyme of the invention can be selectively expressed in the targeted tissues/cells.

Said promoter can be a constitutive promoter or an inducible promoter well known by one skilled in the art. The promoter can be developmentally regulated, inducible or tissue specific.

The invention also relates to a vector comprising a nucleic acid molecule according to the invention. Said vector can be appropriated for semi-stable or stable expression.

The invention also relates to a group of vectors comprising said group of isolated nucleic acid molecules according to the invention.

Particularly said vector according to the invention is a cloning or an expression vector.

The vectors can be viral vectors such as bacteriophages, or non-viral, such as plasmid vectors.

In one embodiment, said vector according to the invention is a bicistronic vector, in particular comprising a nucleic acid molecule according to the invention and a promoter for said catalytic domain of a DNA-dependant RNA polymerase and/or at least one DNA sequence of interest, wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependant RNA polymerase.

Said vector according to the invention can also comprise a promoter for said catalytic domain of a DNA-dependant RNA polymerase.

The invention also relates to a host cell comprising an isolated nucleic acid molecule according to the invention or a group of isolated nucleic acid molecules according to the invention or a vector according to the invention or a group of vectors according to the invention.

The host cell according to the invention can be useful for large-scale protein production.

Preferably, said catalytic domains of the DNA-polymerase RNA polymerase chimeric enzyme according to the invention are from different enzymes than those of the host cell to prevent the competition between the endogenous gene transcription and the transgene transcription.

The invention also relates to a genetically engineered non-human eukaryotic organism, which expresses a chimeric enzyme according to invention. Said non-human eukaryotic organism can be any non-human animals, plants.

The invention also relates to the use, particularly in vitro or ex vivo, of a chimeric enzyme according to the invention or an isolated nucleic acid molecule according to the invention or a group of isolated nucleic acid molecules according to the invention, for the production of RNA molecule with 5′-terminal m⁷GpppN cap.

The invention also relates to the in vitro or ex vivo use of a chimeric enzyme according to the invention or an isolated nucleic acid molecule according to the invention or a group of isolated nucleic acid molecules according to the invention for the production of protein, in particular protein of therapeutic interest like antibody, particularly in eukaryotic systems, such as in vitro synthesized protein assay or cultured cells.

The invention also relates to a method, particularly in vitro or ex vivo, for producing a RNA molecule with 5′-terminal m⁷GpppN cap encoded by a DNA sequence, in a host cell, said method comprising the step of expressing in the host cell a nucleic acid molecule or a group of nucleic acid molecules according to the invention, wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependant RNA polymerase, particularly said promoter being effective in said host cell.

Preferably, said catalytic domain of the DNA-dependent RNA-polymerase of the chimeric enzyme according to the invention is from different enzymes than those of the host cell to prevent the competition between the endogenous gene transcription and said DNA sequence transcription.

In particular, said method according to the invention can further comprise the step of introducing in the host cell said DNA sequence and/or the nucleic acid according to the invention, using well-known methods by one skilled in the art like by transfection using calcium phosphate, by electroporation or by mixing a cationic lipid with DNA to produce liposomes.

In one embodiment, said method according to the invention further comprises the step of inhibiting, in particular silencing (preferably by siRNA) the cellular transcription and post-transcriptional machineries of said host cell.

In one embodiment, said method according to the invention further comprises the step of inhibiting the expression of at least one of the subunits of the endogenous DNA-dependent RNA polymerase and/or of the endogenous capping enzyme in said host cell.

Said additional steps (i e inhibiting, in particular silencing (preferably by siRNA or shRNA) the cellular transcription and post-transcriptional machineries of said host cell and/or inhibiting the expression of one or several subunits of the endogenous DNA-dependent RNA polymerase and/or of the endogenous capping enzyme in said host cell) allow the optimization of RNA molecules with 5′-terminal m⁷GpppN caps synthesis.

As used herein the term “endogenous DNA-dependent RNA polymerase” relates to the endogenous DNA-dependent RNA polymerase of said host cell. When the host cell is a eukaryotic cell, said endogenous DNA-dependent RNA polymerase is the RNA polymerase II.

As used herein the term “endogenous capping enzyme” refers to the endogenous capping enzyme of said host cell.

As used herein the term “inhibiting the expression of a protein” relates to a decrease of at least 20%, particularly at least 35%, at least 50% and more particularly at least 65%, at least 80%, at least 90% of expression of said protein Inhibition of protein expression can be determined by techniques well known to one skilled in the art, including but not limiting to Northern-Blot, Western-Blot, RT-PCR.

The step of inhibiting the expression of the endogenous DNA-dependent RNA polymerase and/or the endogenous capping enzyme in said host cell can be implemented by any techniques well known to one skilled in the art, including but not limiting to siRNA (small interfering RNA) techniques that target said endogenous DNA-dependent RNA polymerase and/or the endogenous capping enzyme, anti-sens RNA techniques that target said endogenous DNA-dependent RNA polymerase and/or the endogenous capping enzyme, shRNA (short hairpin RNA) techniques that target said endogenous DNA-dependent RNA polymerase and/or the endogenous capping enzyme.

In addition to siRNA (or shRNA; short hairpin RNA), other inhibitory sequences might be also considered for the same purpose including DNA or RNA antisense (Liu and Carmichael 1994; Dias and Stein 2002), hammerhead ribozyme (Salehi-Ashtiani and Szostak 2001), hairpin ribozyme (Lian, De Young et al. 1999) or chimeric snRNA U1-antisense targeting sequence (Fortes, Cuevas et al. 2003). In addition, other cellular target genes might be considered for inhibition, including other genes involved in the cellular transcription (e.g. other subunits of the RNA polymerase II or transcription factors), post-transcriptional processing (e.g. other subunit of the capping enzyme, as well as polyadenylation or spliceosome factors), and mRNA nuclear export pathway.

In one embodiment of the method according to the invention, said RNA molecule can encode a polypeptide of therapeutic interest.

In another embodiment, said RNA molecule can be a non-coding RNA molecule chosen in the group comprising siRNA, ribozyme, shRNA and antisense RNA. In particular, said DNA sequence can encode a RNA molecule chosen in the group consisting of mRNA, non-coding RNA, particularly siRNA, ribozyme, shRNA and antisense RNA.

The invention also relates to the use of a chimeric enzyme according to the invention as a capping enzyme and DNA-dependent RNA polymerase.

The invention also relates to a kit for the production of a RNA molecule with 5′-terminal m⁷GpppN cap, comprising at least one chimeric enzyme according to the invention, and/or an isolated nucleic acid molecule and/or a group of nucleic acid molecules according to the invention, and/or a vector according to the invention and/or a group of vectors according to the invention.

In one embodiment the kit of the invention comprises a vector according to the invention and/or a group of vectors according to the invention, wherein said vector(s) comprising:

-   -   a promoter for said catalytic domain of a DNA-dependant RNA         polymerase, and/or     -   at least one DNA sequence of interest, wherein said DNA sequence         is operatively linked to a promoter for said catalytic domain of         a DNA-dependant RNA polymerase.

The kit according to the invention can further comprise:

-   -   a vector comprising a promoter for said catalytic domain of a         DNA-dependant RNA polymerase; and/or     -   at least one DNA sequence of interest, wherein said DNA sequence         is operatively linked to a promoter for said catalytic domain of         a DNA-dependant RNA polymerase.

The invention also relates to a chimeric enzyme according to the invention, an isolated nucleic acid molecule according to the invention, a group of nucleic acid molecules according to the invention or a vector according to the invention, for its use as a medicament, in particular for the prevention and/or treatment of human or animal pathologies, preferably by means of gene therapy.

The invention also relates to a pharmaceutical composition comprising a chimeric enzyme according to the invention, and/or an isolated nucleic acid molecule according to the invention and/or a group of nucleic acid molecules according to the invention, and/or a vector according to the invention. Preferably, said pharmaceutical composition according to the invention is formulated in a pharmaceutical acceptable carrier.

Pharmaceutical acceptable carriers are well known by one skilled in the art.

The pharmaceutical composition according to the invention can further comprise:

-   -   at least one DNA sequence of interest, wherein said DNA sequence         is operatively linked to a promoter for said catalytic domain of         a DNA-dependant RNA polymerase.

Such components (in particular chosen in the group consisting of a chimeric enzyme according to the invention, an isolated nucleic acid molecule according to the invention, a group of isolated nucleic acid molecules according to the invention, a vector according to the invention, a group of vector(s) according to the invention and at least one DNA sequence of interest) can be present in the pharmaceutical composition or medicament according to the invention in a therapeutically amount (active and non-toxic amount).

Such therapeutically amount can be determined by one skilled in the art by routine tests including assessment of the effect of administration of said components on the pathologies and/or disorders which are sought to be prevent and/or to be treated by the administration of said pharmaceutical composition or medicament according to the invention.

For example, such tests can be implemented by analyzing both quantitative and qualitative effect of the administration of different amounts of said aforementioned components (in particular chosen in the group consisting of a chimeric enzyme according to the invention, an isolated nucleic acid molecule according to the invention, a group of isolated molecules according to the invention, a vector according to the invention, a group of vectors according to the invention and at least one DNA sequence of interest) on a set of markers (biological and/or clinical) characteristics of said pathologies and/or of said disorders, in particular from a biological sample of a subject.

The invention also relates to a therapeutic method comprising the administration of a chimeric enzyme according to the invention, and/or an isolated nucleic acid molecule according to the invention, and/or a group of nucleic acid molecules according to the invention and/or a vector according to the invention and/or a group of vectors according to the invention in a therapeutically amount to a subject in need thereof. The therapeutic method according to the invention can further comprise the administration of at least one DNA sequence of interest, wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependant RNA polymerase, in a therapeutically amount to a subject in need thereof.

Said chimeric enzyme, nucleic acid molecule and/or said vector according to the invention can be administrated simultaneously, separately or sequentially of said DNA sequence of interest, in particular before said DNA sequence of interest.

The invention also relates to a pharmaceutical composition according to the invention for its use for the prevention and/or treatment of human or animal pathologies, in particular by means of gene therapy.

Said pathologies can be chosen from the group consisting of pathologies, which can be improved by the administration of said at least one DNA sequence of interest.

The invention also relates to the use of a chimeric enzyme according to the invention, and/or an isolated nucleic acid molecule according to the invention, and/or a group of nucleic acid molecules according to the invention and/or a vector according to the invention, and/or a group of vectors according to the invention for the preparation of a medicament for the prevention and/or treatment of human or animal pathologies, in particular by means of gene therapy.

The invention also relates to a combination product, which comprises:

-   -   at least one enzyme according to the invention and/or at least         one nucleic acid molecule according to the invention and/or a         group of nucleic acid molecules according to the invention         and/or a at least one vector comprising and/or expressing a         nucleic acid molecule according to the invention and/or a group         of nucleic acid molecules according to the invention; and     -   at least one DNA sequence of interest, wherein said DNA sequence         is operatively linked to a promoter for said catalytic domain of         a DNA-dependant RNA polymerase;

for simultaneous, separate or sequential administration.

Said DNA sequence of interest can be an anti-oncogene (a tumor suppressor gene).

Said DNA sequence of interest can encode a polypeptide of therapeutic interest or a non-coding RNA chosen in the group comprising siRNA, ribozyme, shRNA and antisense RNA.

Said polypeptide of therapeutic interest can be selected from, a cytokine, a cell or nuclear receptor, a ligand, a coagulation factor, the CFTR protein, insulin, dystrophin, a growth hormone, an enzyme, an enzyme inhibitor, a polypeptide which has an antineoplastic effect, a polypeptide which is capable of inhibiting a bacterial, parasitic or viral, in particular HIV, infection, an antibody, a toxin, an immunotoxin, a subunit of RNA polymerase II (in particular the Rpb1subunit of RNA polymerase II, which can be inhibited by the alpha-amanitin toxin) and a marker.

Preferably, the combination product according to the invention can be formulated in a pharmaceutical acceptable carrier.

In one embodiment of the combination product according to the invention, said vector is administrated before said DNA sequence of interest.

The invention also relates to a combination product according to the invention, for its use for the prevention and/or treatment of human or animal pathologies, in particular by means of gene therapy.

Said pathologies can be chosen from the group consisting of pathologies, which can be improved by the administration of at least one DNA sequence of interest, as described above.

For example, said pathologies, as well as their clinical, biological or genetic subtypes, can be chosen from the group comprising cancers and their predisposition (especially breast and colorectal cancers, melanoma), malignant hemopathies (in particular leukemias, Hodgkin's and non-Hodgkin's lymphomas, myeloma), coagulation and primary hemostasis disorders, hemoglobinopathies (especially sickle cell anemia and thalassemias), autoimmune disorders (including systemic lupus erythematosus and scleroderma), cardiovascular pathologies (in particular cardiac rhythm and conduction disorders, and hypertrophic cardiomyopathy), metabolic disorders (especially type I and type II diabetes mellitus and their complications, dsylipidemia, atherosclerosis and their complications, mucopolysaccharidoses, glycogen storage diseases, phenylketonuria), infectious disorders (including AIDS, viral hepatitis B, viral hepatitis C, influenza flu and other viral diseases; botulism, tetanus and other bacterial disorders; malaria and other parasitic disorders), muscular disorders (including Duchenne muscular dystrophy and Steinert myotonic muscular dystrophy), respiratory diseases (especially cystic fibrosis and alpha-1 antitrypsin deficiency), renal disease (especially polycystic kidney disease), liver diseases (including cirrhosis, Wilson disease, hepatotoxicity due to the alpha-amanitin, drug-related hepatotoxicity), colorectal disorders (including Crohn's disease and ulcerative colitis), ocular disorders especially retinal diseases (especially Leber's amaurosis, retinitis pigmentosa, age related macular degeneration), central nervous system disorders (especially Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, neurofibromatosis, adrenoleukodystrophy, bipolar disease, schizophrenia and autism), and skin and connective tissue disorders (especially Marfan syndrome and psoriasis).

In one embodiment, the combination product of the invention comprises:

-   -   at least one vector comprising and expressing a nucleic acid         molecule according to the invention and/or a group of nucleic         acid molecules according to the invention, wherein said         catalytic domain of a DNA-dependant RNA polymerase is a         catalytic domain of a bacteriophage DNA-dependant RNA         polymerase, particularly of a T7 bacteriophage DNA-dependant RNA         polymerase; and     -   at least one DNA sequence of interest, wherein said DNA sequence         is operatively linked to a promoter for said catalytic domain of         a bacteriophage DNA-dependant RNA polymerase, particularly of a         T7 bacteriophage DNA-dependant RNA polymerase, wherein said DNA         sequence of interest encodes the Rpb1 subunit of RNA polymerase         II, which can be inhibited by the alpha-amanitin toxin.

The invention also relates to this combination product for its use for the prevention and/or treatment of human or animal hepatotoxicity due to the alpha-amanitin, by means of gene therapy.

The invention also relates to a method for producing the chimeric enzyme according to the invention comprising the step of expressing in at least one host cell said nucleic acid molecule or said group of nucleic acid molecules encoding the chimeric enzyme of the invention in conditions allowing the expression of said nucleic acid molecule(s) in said host cell.

The invention also relates to a method for producing the chimeric enzyme according to the invention comprising the steps of:

-   -   expressing a part of said group of nucleic acid molecules         encoding a chimeric enzyme of the invention in a first host cell         in conditions allowing the expression of said nucleic acid         molecules in said host cell, to obtain a first part of the         chimeric enzyme of the invention;     -   expressing the other part of said group of nucleic acid         molecules encoding the chimeric enzyme of the invention in a         second host cell in conditions allowing the expression of said         nucleic acid molecules in said host cell to obtain a second part         of the chimeric enzyme of the invention; and     -   assembling said first part and said second part to obtain the         chimeric enzyme of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 represents a firefly luciferase gene reporter expression assay, which was used to monitor the translation yields triggered by a chimeric enzyme according to the invention the NP868R-T7RNAP. The pNP868R-T7RNAP or the pT7RNAP plasmids were co-transfected with the pT7p-Luciferase plasmid in the human HEK-293 cultured cells. The expression of NP868R-T7RNAP and T7RNAP enzymes is driven by the RNA polymerase II-dependent CMV promoter of the corresponding plasmids. The NP868R-T7RNAP and T7RNAP enzymes, in turn, are expected to initiate transcription at the T7 promoter of the pT7p-Luciferase gene reporter plasmid. If both the mRNA capping and DNA-dependent RNAP enzymatic activities of the NP868R-T7RNAP enzymes are retained, luciferase mRNA having a m⁷GpppGm cap structures are to be synthesized, which can be translated into firefly luciferase protein and detected by cell luminescence assay. In contrast, the T7RNAP enzyme is expected to synthesize RNA molecules without 5′-terminal m⁷GpppN cap, which are therefore poorly translated.

FIG. 2 (A-B) represents the physical maps of the pNP868R-T7RNAP and pT7RNAP plasmids. Physical maps of (A) pT7RNAP plasmid, which encodes for the wild-type phage DNA-dependent T7 RNA polymerase, (B) pNP868R-T7RNAP plasmids, which encodes for a fusion between the NP868R mRNA capping enzymes (African Swine Fever Virus) and the wild-type phage DNA-dependent T7 RNA polymerase (bacteriophage T7), via a flexible (Gly₃Ser)₄ linker. These two plasmids have the same design: CMV promoter, Kozak sequence followed by the NP868R-T7RNAP or T7RNAP open-reading frames (ORFs), poly[A]-track, TΦ terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal.

FIG. 3 (A-C) represents the physical maps of the firefly luciferase gene reporter plasmids. (A) pT7p-Luciferase: was designed to assay the activity of the NP868R-T7RNAP and the T7RNAP enzymes. It consist of an array of RNA polymerase promoters (T7, T3 and SP6 phage RNAP promoters, followed by the E. coli ribosomal rrnD1 promoter), a Lac operator sequence, the entire ORF of the firefly luciferase, a poly[A]-track, a hepatitis-D ribozyme encoding sequence for RNA auto-cleavage and the TΦ terminator in pET-22b(+) backbone, (B) BamHI-digested pT7p-Luciferase: in which the physical connection between the luciferase ORF and the promoter array is disrupted by the restriction enzyme digestion. This plasmid is used as a negative control. Arrows indicate the sites of digestion. (C) pCMV-Luciferase: in which the entire ORF of the firefly luciferase is under control of the CMV promoter. This plasmid is used as a positive comparator.

FIG. 4 (A-C) shows the Firefly luciferase gene reporter expression after plasmid transfection in HEK-293 cells. HEK-293 cells were cultured and transfected as described above. Cells were transfected with either the pNP868R-T7RNAP or the pT7RNAP plasmids (0.4 μg DNA/well and 1 μL/well Lipofectamine 2000), and/or the pT7p-Luciferase, BamHI-digested pT7p-Luciferase, or pCMV-T7RNAP (0.4 μg DNA/well and 1 μL/well Lipofectamine 2000), or none. The firefly luciferase luminescence was assayed at selected time points using the Luciferase Assay System (Promega, Madison Wis. USA). To normalize for transfection efficiency, cells were also transfected with the pORF-eSEAP plasmid, which encodes for the secreted placental alkaline phosphatase (SEAP) that was assayed in cell culture medium using the Quanti-Blue colorimetric enzyme assay kit (InvivoGen, San Diego, Calif.). Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU; relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Two independent repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM). Statistical analyses were performed using Student's t two-tailed test. (A) Firefly luciferase gene reporter expression in pNP868R-T7RNAP/pT7p-Luciferase, pT7RNAP/pT7p-Luciferase and pCMV-Luciferase transfected cells. Cells transfected with the pNP868R-T7RNAP/pT7p-Luciferase and the pCMV-Luciferase plasmids display 23- and 33-fold fold higher signal than the cells co-transfected with pT7RNAP/pT7p-Luciferase, respectively (*p<0.05). (B) Firefly luciferase gene reporter expression for cells transfected with pT7RNAP/pT7p-Luciferase, pT7RNAP/BamHI-digested pT7p-Luciferase (*p<0.05) and other control conditions (pT7RNAP alone, pT7p-Luciferase digested or not alone, or transfection reagent only). (C) Firefly luciferase gene reporter expression for the pNP868R-T7RNAP/pT7p-Luciferase, pNP868R-T7RNAP/BamHI-digested pT7p-Luciferase (*p<0.05) and other control conditions (pNP868R-T7RNAP alone, pT7p-Luciferase digested or not alone, or transfection reagent only).

FIG. 5 (A-C) represents the Firefly luciferase gene reporter expression of HEK-293 transfected cells treated with α-amanitin. (A) Schematic diagram of the assay. For the pCMV-Luciferase plasmid (expression of luciferase is driven by the RNA polymerase II-dependent CMV promoter), α-amanitin was added to cell medium (at 0 or 20 μg/ml) simultaneously to cell transfection. For the pNP868R-T7RNAP/pT7p-Luciferase plasmids, a first transfection with the pNP868R-T7RNAP plasmid (expression of NP868R-T7RNAP is driven by the RNA polymerase II-dependent CMV promoter) was performed 24 hours before addition of α-amanitin to the cell medium (at concentrations ranging from 0 to 20 μg/ml) and a second transfection with the pT7p-Luciferase plasmid. Two repetitions of these experiments were performed. Errors bars represent standard error of the mean (SEM). Statistical analysis was performed as described above. (B) α-amanitin nearly completely abolished luciferase gene reporter expression of cells transfected with the pCMV-Luciferase plasmid; *p<0.05) (C) α-amanitin triggered only a mild decrease of luciferase expression signal in cells transfected with the pNP868R-T7RNAP/pT7p-Luciferase plasmids (*p<0.05), which therefore suggest that the transcription by the NP868R-T7RNAP enzyme is dependent of its phage DNA-dependent T7 RNA polymerase moiety.

FIG. 6 (A-C) represents the cell viability, cytotoxicity and apoptosis assays of HEK-293 transfected cells. Cells were cultured and transfected as above with the pNP868R-T7RNAP or pT7RNAP plasmids. Cell viability, cytotoxicity and apoptosis were assessed at selected time points using the ApoTox-Glo Triplex Assay. Two repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM). Cell viability, cytotoxicity and apoptosis levels were expressed as the luminescence/fluorescence signal in studied cells subtracted for luminescence/fluorescence in untreated cells. Statistical analysis was performed as above. The transfection reagent (i.e. Lipofectamine 2000), with or without plasmid DNA, impairs cell viability, cytotoxicity and apoptosis. However, no statistically significant difference was observed between cells transfected with the pNP868R-T7RNAP plasmid and the pT7RNAP plasmid for (A) cell viability levels, at all time points, except at day 1 (two-tailed Student's t test, *p<0.05), (B) cytotoxicity levels at all time points, or (C) apoptosis levels at all time points.

FIG. 7 (A-D) represents the physical maps of the plasmids used for the monomeric NP868R-T3RNAP and NP868R-SP6RNAP chimeric enzymes. Physical maps of: (A) pT3RNAP plasmid, which encodes for the phage DNA-dependent T3 RNA polymerase, (B) pSP6 RNAP plasmid, which encodes for the phage DNA-dependent SP6 RNA polymerase, (C) pNP868R-T3RNAP plasmid, which encodes for a fusion between the NP868R African Swine Fever Virus mRNA capping enzyme and the phage DNA-dependent T3 RNA polymerase, via the flexible (Gly₃Ser)₄ linker, (D) pNP868R-SP6RNAP plasmid, which encodes for a fusion between the NP868R African Swine Fever Virus mRNA capping enzyme and the phage DNA-dependent SP6 RNA polymerase, via the flexible (Gly₃Ser)₄ linker. These two plasmids have the same design: CMV promoter, Kozak sequence followed by the ORFs, poly[A]-track, TΦ terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal.

FIG. 8 represents the expression of the luciferase reporter gene by the monomeric NP868R-T3RNAP and NP868R-SP6RNAP chimeric enzymes. Transfection and luciferase assay were performed as previously described. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Four repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM).

FIG. 9 (A-C) represents the schematic structure of heterodimeric and heterotrimeric chimeric enzymes. (A) Heterodimeric RR₁₂₃₄L-NP868R/EE₁₂₃₄L-T7RNAP enzyme. The leucine zippers EE₁₂₃₄L (acid) and RR₁₂₃₄L (basic) were added to the amino-terminal ends of NP868R and T7 RNA polymerase, respectively, which interact to form an heterodimer; (B) Heterodimeric D12L/D1R-T7RNAP CCPP enzyme. The D1R subunit of the vaccinia virus mRNA capping enzyme is fused to the amino-terminal end of the single-unit T7 RNA polymerase. When co-expressed with the D12L subunit of the mRNA capping enzyme, D1R-T7RNAP forms a heterodimer designated D12L/D1R-T7RNAP. For simplification, the other construction, i.e. D1R/D12L-T7RNAP chimeric enzyme, is not shown; (C) Heterotrimeric D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP chimeric enzyme. The leucine zippers EE₁₂₃₄L (acid) and RR₁₂₃₄L (basic) were added to the amino-terminal ends of D1R and T7 RNA polymerase, respectively. The co-expression of RR₁₂₃₄L-D1R together with EE₁₂₃₄L-T7RNAP and the D12L subunit of the vaccinia virus mRNA capping enzyme form a heterotrimer. For simplification, the other construction, i.e. D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP chimeric enzyme, is not shown. Open arrows indicate leucine-zipper binding in antiparallel orientation. Black arrows indicate other types of protein interaction.

FIG. 10 (A-H) represents physical maps of the plasmids used for heterodimeric and heterotrimeric chimeric enzymes. Physical maps of (A) pD1R plasmid, which encodes for D1R, the large subunit of the vaccinia mRNA capping enzyme, (B) pD12L plasmid, which encodes for the D12L, the small subunit of the vaccinia mRNA capping enzyme, (C) pRR₁₂₃₄L-NP868R plasmid, which encodes for the RR₁₂₃₄L leucine-zipper fused to the amino-terminal end of NP868R, the African Swine Fever Virus mRNA capping enzyme, (D) pRR₁₂₃₄L-D1R plasmid, which encodes for the RR₁₂₃₄L leucine-zipper fused to the amino-terminal end of D1R, the large subunit of the vaccinia mRNA capping enzyme, (E) pRR₁₂₃₄L-D12L plasmid, which encodes for the RR₁₂₃₄L leucine-zipper fused to the amino-terminal end of D12L, the small subunit of the vaccinia mRNA capping enzyme, (F) pEE₁₂₃₄L-T7RNA plasmid, which encodes for the pEE₁₂₃₄L leucine-zipper fused to the phage DNA-dependent T7 RNA polymerase, (G) pD1R-T7RNAP plasmid, which encodes for a fusion between the large subunit of the vaccinia mRNA capping enzyme and the phage DNA-dependent T7 RNA polymerase, via the flexible (Gly₃Ser)₄ linker, (H) pD12L-T7RNAP plasmid, which encodes for a fusion between the small subunit of the vaccinia mRNA capping enzyme and the phage DNA-dependent T7 RNA polymerase, via the flexible (Gly₃Ser)₄ linker. All these plasmids have the same design: CMV promoter, Kozak sequence followed by the ORFs, poly[A]-track, TΦ terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal.

FIG. 11 represents the expression of the luciferase reporter gene by the heterodimeric RR₁₂₃₄L-NP868R/EE₁₂₃₄L-T7RNAP chimeric enzymes. HEK-293 cells were cultured and transfected as described above. Cells were transfected with either the corresponding plasmids (0.4 μg DNA/well and 1 μL/well lipofectamine 2000) and pT7p-Luciferase, or pCMV-T7RNAP (0.4 μg DNA/well and 1 μL/well lipofectamine 2000). Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Four repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM).

FIG. 12 represents the expression of the luciferase reporter gene by the heterodimeric D1R/D12L-T7RNAP and D12L/D1R-T7RNAP chimeric enzymes. Transfection and luciferase assay were performed as previously described. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Two repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM).

FIG. 13 represents the expression of the luciferase reporter gene by the heterotrimeric D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP and D12L/EE₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP chimeric enzymes. Transfection and luciferase assay were performed as previously described. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Two repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM).

FIG. 14 represents the expression of the luciferase reporter gene by the monomeric NP868R-SP6RNAP chimeric enzymes in presence of siRNAs targeting the large subunit of RNA polymerase II (POL2AR) or the human capping enzyme (RNGTT). Transfection and luciferase assay were performed as previously described, except that siRNA at 25 nM final concentration were added to the transfection reagent. The siRNA SI04364381, SI04369344, SI04250162 and SI04354420 target the POLR2A gene, whereas the siRNA SI00055986, SI03021508, SI00055972, and SI00055979 target RNGTT. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Two repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM).

FIG. 15 represents the dose effect activity of siRNAs targeting the large subunit of RNA polymerase II (POL2AR) and the human capping enzyme (RNGTT). Transfection and luciferase assay were performed as previously described, except that siRNA were added at concentration ranging from 0 to 100 nM to the transfection reagent. The siRNA SI04369344 and SI00055972 target the POLR2A and RNGTT genes, respectively. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Three repetitions of this experiment were performed. Errors bars represent standard error of the mean (SEM).

The present invention will be explained in detail with examples in the following, but the technical scope of the present invention is not limited to these examples.

EXAMPLES Example 1 Example of Active Monomeric Chimeric Enzyme Np868R-T7RNAP

I. Plasmids

One plasmid has been synthesized, which encode for fusions between NP868R, the mRNA capping enzyme of the African Swine Fever Virus, and the wild-type phage DNA-dependent RNA polymerase of the bacteriophage T7. The capping enzyme was fused to the amino-terminal end of the T7 RNA polymerase via a (Gly₃Ser)₄ linker. The pNP868R-T7RNAP plasmid was used to assess the activity of the encoded enzyme by a firefly luciferase gene reporter expression assay (FIG. 1). In brief, pNP868R-T7RNAP and pT7p-Luciferase plasmids were co-transfected in the human HEK-293 cultured cells. The expression of the NP868R-T7RNAP enzyme is driven by the RNA polymerase II-dependent CMV promoter of the corresponding plasmid. NP868R-T7RNAP enzyme, in turn, is expected to initiate the transcription of the pT7p-Luciferase plasmid at its T7 promoter. If both the mRNA capping and DNA-dependent RNAP enzymatic activities of the NP868R-T7RNAP enzyme are retained, luciferase mRNA having m⁷GpppGm cap structures are to be synthesized, which in turn can be translated into firefly luciferase protein and detected by cell luminescence assay. In addition, the pNP868R-T7RNAP plasmid was used to investigate the cellular distribution of the enzyme, as well as the cell viability, cytotoxicity and apoptosis related with the expression of the enzyme in the HEK-293 cells.

The plasmid encoding for the NP868R-T7RNAP and T7RNAP (T7 RNA polymerase) enzymes were synthesized in four steps by GeneArt AG (Regensburg, Germany). The protein sequence encoded by pT7RNA plasmid corresponds to SEQ ID NO:19. The protein sequence encoded by pNP868R-T7RNAP plasmid corresponds to SEQ ID NO:20. Firstly, a DNA fragment containing the T7 RNA polymerase promoter and the multiple cloning site (MCS) was removed from the pCMV-Script plasmid (Stratagene, La Jolla, Calif. USA). Secondly, a cassette was introduced in the pCMV-Script plasmid between its CMV promoter and its SV40 polyadenylation signal. This cassette consisted of the Lac operator stem-loop (Gilbert and Maxam 1973), a MCS, a poly[A]-tract, and a TΦ class-I hairpin terminator signal (Lyakhov, He et al. 1997). Thirdly, the Kozak consensus sequence for initiation of translation (Kozak 1987), followed by the entire open-reading frame (ORF) of the NP868R-T7RNAP or T7RNAP enzymes were assembled from synthetic oligonucleotides using a PCR-based method, cloned and fully sequence verified. The ORF of the NP868R-T7RNAP (SEQ ID NO:21) and of the ORF of the T7RNAP (SEQ ID NO:22) were optimized by altering for preferred codon usage, removing of cis-acting elements such as cryptic splice sites and poly(A) signals, as well as improving mRNA stability by removal of direct repeats and secondary structure elements. Fourthly, the entire ORFs of each NP868R-T7RNAP or T7RNAP were subcloned in the MCS of the cassette, resulting in the pNP868R-T7RNAP plasmid and the pT7RNAP plasmid. As a consequence of the construction strategy, two additional amino-acids (Glu, Phe) were added immediately downstream to the ATG of the Kozak sequence, two other were added immediately upstream to the (Gly₃Ser)₄ linker (Gly, Pro), and two immediately downstream to this linker (Leu, Glu) of the NP868R-T7RNAP enzyme. Finally, the pNP868R-T7RNAP and pT7RNAP plasmids had the following design (FIGS. 2A and 2B): CMV promoter, Kozak sequence followed by the NP868R-T7RNAP or T7RNAP ORFs, poly[A]-track, TΦ terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal.

Two plasmids encoding for the firefly luciferase reporter gene were synthesized by Eurofins/MWG/Operon (Ebersberg, Germany). The pET-22b(+)RNAPp-Luciferase plasmid (named pT7p-Luciferase thereafter) was designed to assay the activity of the chimeric enzyme according to the invention. A test sequence was introduced in the pET-22b(+) backbone (Novagen, San Diego, Calif. USA), which consisted of an array of RNA polymerase promoters (T7, T3 and SP6 phage RNAP promoters, followed by the E. coli ribosomal rrnD1 promoter), a Lac operator stem-loop sequence, the entire ORF of the firefly luciferase, a poly[A]-track, a hepatitis-D ribozyme encoding sequence for RNA auto-cleavage (Conzelmann and Schnell 1994; Garcin, Pelet et al. 1995; Bridgen and Elliott 1996; Schurer, Lang et al. 2002; Walker, Avis et al. 2003) and the TΦ terminator for phage RNA polymerase transcription (FIG. 3A). A BamHI-digested version of the pT7p-Luciferase plasmid, which disrupts the physical connection between the luciferase ORF and the T7 promoter, was also used as negative control (FIG. 3B). Moreover, the pCMV-Luciferase plasmid, which was used as an active comparator, contained the firefly luciferase downstream to the RNA polymerase II-dependent CMV promoter of the pCMV-Script plasmid (FIG. 3C).

II. Cell Culture and Transfection

The Human Embryonic Kidney 293 cells (HEK-293, ATCC CRL 1573) were grown at 37° C. with 5% CO2. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 3.97 mM L-alanyl-L-glutamine (substituted on a molar equivalent basis for L-glutamine), 10% fetal bovine serum (FBS), 1% non-essential amino-acids, 1% penicillin and streptomycin, and 0.2% fungizone.

The day before transfection, HEK-293 cells were plated in 24 well plates at densities of approximately 8×10⁴ cells per well. One hour prior to transfection, the medium was changed to fresh complete medium without antibiotics. Transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif. USA) according to manufacturer's recommendations. In brief, plasmid DNA diluted in Opti-MEM I reduced serum medium (Invitrogen, Carlsbad, Calif. USA) and mixed with Lipofectamine 2000, were added to the cell medium. Following transfection, cells were incubated up to 120 hours prior to testing for luciferase and SEAP gene reporter expression.

Cells were co-transfected with the pT7RNAP or pNP868R-T7RNAP (0.4 μg DNA/well and 1 μL/well Lipofectamine 2000), together with the pT7p-Luciferase reporter plasmid (0.4 μg DNA/well and 1 μL/well Lipofectamine 2000). A series of other transfection conditions were used as negative controls and included: (a) the same co-transfection as before, except that the pT7p-Luciferase was digested by the BamHI restriction enzyme, which disrupts the physical connection between the luciferase ORF and the T7 promoter, (b) the pNP868R-T7RNAP or pT7RNAP plasmids alone, (c) the pT7p-Luciferase reporter plasmid digested or not alone, (d) the transfection reagent alone (i.e. Lipofectamine 2000). Cells were also transfected with the pORF-eSEAP plasmid (InvivoGen, San Diego, Calif.; used to normalize for transfection efficacy), as well as with the pCMV-T7RNAP plasmid (used as an active comparator).

III. Firefly Luciferase Luminescence and SEAP Colorimetric Assays

The firefly luciferase luminescence was assayed with the Luciferase Assay System according to manufacturer's recommendations (Promega, Madison Wis. USA). In brief, HEK-293 cells were lysed in Cell Culture Lysis Reagent (CCLR) lysis buffer, and then centrifuged at 12,000×g for 2 minutes at 4° C. Luciferase Assay Reagent (Promega; 100 μl/well) was added to supernatant (20 μl/well). Luminescence readout was taken on a luminometer reader (Fluostar; BMG Labtech, Offenburg Germany) according to the manufacturer's instructions.

The expression of pORF-eSEAP plasmid was used to normalize for transfection efficiency. This plasmid encodes for the secreted placental alkaline phosphatase (SEAP), which was assayed for enzymatic activity in cell culture medium using the Quanti-Blue colorimetric enzyme assay kit (InvivoGen, San Diego, Calif.) at selected time points. Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU; relative light units), then divided by SEAP absorbance to normalize for transfection efficacy (OD, optic density) ratio.

IV. Statistical Analysis

All statistical analyses were performed using Student's t two-tailed test adjusted by Holm-Bonferroni correction for multiple testing, if appropriate. A p-value of less than 0.05 was regarded as being significant.

V. Gene Reporter Expression Assay

A firefly luciferase reporter luminescence assay was used to assess the translatability of mRNA generated by the chimeric enzyme according to the invention or T7RNAP enzyme. The co-transfection of the pT7RNAP and pT7p-Luciferase plasmids triggered low but detectable luciferase expression signal in comparison to cells co-transfected with the pT7RNAP/BamHI-digested version of the pT7p-Luciferase plasmid (which therefore demonstrate that luciferase gene reporter expression is driven by the phage T7 promoter; FIGS. 4A and 4B). This is in agreement with previously published reports, which have shown that the T7RNAP expressed in eukaryotic cells can synthesize RNA molecules which are poorly translated because of their absence of 5′-capping (Fuerst, Niles et al. 1986; Chen, Li et al. 1994). A drastic reduction of the firefly luciferase gene expression signal was also observed when the transfection was performed without the pT7RNAP plasmid (which confirm that luciferase expression require the presence of T7RNAP) or the pT7p-Luciferase plasmid (which confirm the specificity of the luminescence signal), or both.

The pNP868R-T7RNAP plasmid was cotransfected with the pT7p-Luciferase plasmid and tested under same conditions as above. At peak, approximately 23-fold higher luciferase expression signal was observed with pNP868R-T7RNAP/pT7p-Luciferase than with the pT7RNAP/pT7p-Luciferase plasmids (FIG. 4A). The specificity of the above findings was confirmed by the co-transfection of BamHI-digested version of the pT7p-Luciferase plasmid, as well as the transfection by pNP868R-T7RNAP or pT7p-Luciferase plasmids digested or not alone, which gave drastically reduced luciferase expression signal (FIG. 4C). At peak, co-transfection of pNP868R-T7RNAP/pT7p-Luciferase plasmids gave 72% of the luciferase expression signal to that of pCMV-T7RNAP plasmid (FIG. 4A).

In summary, the activity of the chimeric NP868R-T7RNAP enzyme according to the invention encoded by the pNP868R-T7RNAP plasmid has been demonstrated using a firefly luciferase reporter luminescence assay. The specificity of the present findings is supported by a series of controls, which suggest that both the mRNA capping and DNA-dependent RNA polymerase enzymatic activities of the NP868R-T7RNAP enzyme are retained when expressed in HEK-293 cells.

VI. Gene Reporter Expression Assay in Alpha-Amanitin Treated Cells

To further demonstrate that the transcription by pNP868R-T7RNAP is dependent of its phage DNA-dependent T7 RNA polymerase moiety, gene transfection assays were also performed in α-amanitin treated cells. Alpha-amanitin is a specific inhibitor of the nuclear RNA polymerase II (Jacob, Sajdel et al. 1970; Kedinger, Gniazdowski et al. 1970; Lindell, Weinberg et al. 1970), which binds its Rpb1 subunit (Bushnell, Cramer et al. 2002). In contrast, alpha-amanitin has no effect on transcription by the phage T7 RNA polymerase which was used to engineer the NP868R-T7RNAP chimeric enzyme according to the invention (Kupper, McAllister et al. 1973; Engleka, Lewis et al. 1998).

To initiate the expression of the NP868R-T7RNAP enzyme, which is driven by the RNA polymerase II-dependent CMV promoter, cells were transfected with the pNP868R-T7RNAP 24 hours before addition of α-amanitin to cell medium (at concentrations ranging from 0 to 20ηg/ml) and a second transfection with the pT7p-Luciferase plasmid (FIG. 5A). For the pCMV-Luciferase plasmid, cells were simultaneously transfected and treated with α-amanitin (at 0 or 20 μg/ml; FIG. 5A). Gene reporter expression was expressed as the luciferase luminescence in studied cells subtracted for luminescence in cells treated with the transfection reagent only (RLU; relative light units), then divided by SEAP absorbance to normalize for transfection efficacy (OD, optic density) ratio.

As expected, α-amanitin nearly completely abolished firefly luciferase gene reporter expression of pCMV-Luciferase transfected cells (FIG. 5B). In contrast, only a mild decrease of luciferase expression was triggered by α-amanitin at all concentrations in pNP868R-T7RNAP/pT7p-Luciferase transfected cells (FIG. 5C).

The present findings, therefore confirms that the transcription by NP868R-T7RNAP enzyme depends of the enzymatic activity of its phage T7 DNA-dependent RNA polymerase moiety.

VII. Immunofluorescence

The subcellular distribution of the NP868R-T7RNAP enzyme was investigated by indirect immunofluorescence. HEK-293 cells were plated in 24 well plates at 8×10⁴ cells/well, on poly-L-lysine coated coverslips (BD BioCoat; Bioscience, Mississauga, ON USA), then transfected as previously described. Six and 24-hours after transfection, cells were washed in phosphate buffered saline (PBS), and then fixed in 4% paraformaldehyde for 15 minutes. After fixation, cells were washed with PBS, and then permeabilised for 30 minutes in PBS containing 5% goat serum (Invitrogen), 0.1% Triton X-100 and 0.02% sodium azide. Cells were incubated overnight at 4° C. with the mouse monoclonal antibody raised against T7 RNA Polymerase (1:200, Novagen). After extensive washing with PBS, cells were incubated for 3 hours at room temperature with fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse IgG (Sigma-Aldrich). Cell nuclei were stained with 4′-6-Diamidino-2-phenylindole (DAPI) for 5 minutes. Cells were then washed and mounted in the anti-fade medium Mowiol 4-88 (Calbiochem, Gibbstown, N.J. USA). Cells were analyzed by using an epifluorescence microscope with appropriate filters.

As expected, a weak but detectable FITC signal was observed at both 6 and 24-hours in the cytoplasm of cells transfected with the pNP868R-T7RNAP plasmid, while their nuclei were stained by DAPI.

VIII. Cell Viability, Cytotoxicity and Apoptosis Assays

The ApoTox-Glo Triplex Assay (Promega, Madison Wis.) was used to investigate whether the expression of the NP868R-T7RNAP enzyme impair viability, or induce toxicity or apoptosis of transfected cells. Two protease activities were assayed by fluorescence: one is a marker of cell viability (i.e. the peptide substrate GF-AFC), and the other is a marker of cytotoxicity (i.e. the peptide substrate bis-AAF-R110). Apoptosis was assayed by the luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity.

Cell culture and transfections were performed as previously, except that HEK-293 cells were plated in 96-well plates at densities ranging of 1.2×10⁴ cells per well. Cells were transfected with the pNP868R-T7RNAP plasmid, the pT7RNAP plasmid, or the transfection reagent only. ApoTox-Glo Triplex Assay was performed according to manufacturer's recommendations. In brief, at selected time points, the viability/cytotoxicity reagent containing both GF-AFC Substrate and bis-AAF-R110 substrates were added to the wells and incubated for 30 minutes at 37° C., before fluorescence assessment at two different wavelength sets for viability and cytotoxicity. The caspase reagent was then added to all wells, and luminescence was measured after 30 minutes incubation at room temperature. Statistical analysis was performed as above. Cell viability, cytotoxicity and apoptosis levels were expressed as the luminescence/fluorescence signal in studied cells subtracted for luminescence/fluorescence in untreated cells.

As previously reported (Patil, Rhodes et al. 2004), the cell viability, cytotoxicity and apoptosis were significantly impaired in cells treated with the transfection reagent (i.e. Lipofectamine 2000) as compared to untreated cells (FIGS. 6A, 6B and 6C). As also expected, cell viability, cytotoxicity and apoptosis were generally more impaired when plasmid DNA were added to transfection mixture (FIGS. 6A, 6B and 6C). However, at all studied time points, the cell viability, cytotoxicity or apoptosis markers of cells transfected with the pNP868R-T7RNAP plasmid were not statistically different to that of pT7RNAP plasmid, except 24 hours after transfection for cell viability only, which is possibly due to hazard only (FIGS. 6A, 6B and 6C; two-tailed Student's t test for individual time points, P-value<0.05).

In conclusion, no obvious difference in cytotoxicity, cell viability, and apoptosis of the NP868R-T7RNAP enzyme can be demonstrated in comparison to T7RNAP, which has no recognized capping enzymatic activity.

Example 2 Examples of Active Monomeric Chimeric ENZYMES NP868R-T3RNAP and NP868R-SP6RNAP

Two other types of monomeric chimeric enzymes according to the invention have been generated, which consist of NP868R, the monomeric mRNA capping enzyme of the African Swine Fever Virus, fused to the amino-terminal end of the wild type T3 or SP6 monomeric bacteriophage DNA-dependent RNA polymerases, via the flexible linker (Gly₃Ser)₄.

I. Methods

The sequences used to generate said monomeric chimeric enzymes were assembled from synthetic oligonucleotides using a PCR-based method, cloned and fully sequence verified. These sequences were subcloned in the pCMV-Script plasmid containing the subcloning cassette previously described. Finally, all the plasmids used for expression had the similar design: CMV IE1 promoter/enhancer promoter, Kozak sequence followed by the ORFs, poly[A]-track, TΦ terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal (FIG. 7 (A-D)).

As a consequence of the subcloning strategy, amino-acids were added immediately downstream to the ATG of the Kozak sequence encoded by the plasmids (Glu-Phe-Leu-Glu for pT3RNAP and pSP6RNAP; Glu-Phe for pNP868R-T3RNAP and pNP868R-SP6RNAP). In addition, two amino-acids were added immediately upstream (Gly-Pro for pNP868R-T3RNAP and pNP868R-SP6RNAP) or downstream to the (Gly₃Ser)₄ linker (Leu-Glu for pNP868R-T3RNAP and pNP868R-SP6RNAP).

HEK-293 cells were grown as previously described in 24-wells plates and transfected using the Lipofectamine 2000 reagent, and the appropriate plasmids (0.4 μg DNA/well, plus 1 μL/well lipofectamine 2000, per transfected plasmid). The firefly luciferase luminescence was assayed as previously described using the pT7p-Luciferase (which also contains both the T3 and SP6 promoters) and the Luciferase Assay System. The expression of pORF-eSEAP plasmid was used to normalize for transfection efficacy as previously described. Statistical analyses were performed using Student's t two-tailed test adjusted by Holm-Bonferroni correction for multiple testing, if appropriate. A p-value of less than 0.05 was regarded as being statistically significant.

II. Results

As shown in FIG. 8, when co-transfected with the reporter pT7p-luciferase plasmid, both pNP868R-T3RNAP and NP868R-SP6RNAP show strong luciferase gene reporter signal, which was 14- and 56-folds higher than pT3RNAP or pSP6RNAP, respectively (p<0.001 for each comparisons, Student's t-test). The NP868R-T3RNAP enzyme has 36% activity to that of pCMV-T7RNAP plasmid (p<0.001, Student's t-test), whereas or NP868R-SP6RNAP heterodimeric enzyme shows 1.1-fold luciferase reporter gene expression to that of pCMV-T7RNAP plasmid (non-statistically significant difference, Student's t-test).

These results demonstrate the activity of different types of monomeric chimeric enzymes according to the invention.

Example 3 Examples of Active Dimeric and Trimeric Chimeric Enzymes

Different types of active oligomeric chimeric enzymes according to the invention have been generated as shown in FIG. 9:

-   -   one heterodimeric chimeric enzyme, resulting of the non-covalent         linkage between the monomeric African Swine Fever Virus mRNA         capping enzyme NP868R and the monomeric T7 RNA polymerase, via         the EE₁₂₃₄L and RR₁₂₃₄L leucine-zippers, which form the         heterodimeric RR₁₂₃₄L-pNP868R/EE₁₂₃₄L-T7RNAP chimeric enzyme,     -   two heterodimeric chimeric enzymes obtained by fusion between         each of the two subunits of the vaccinia virus mRNA capping         enzyme (i.e. D1R or D12L) with the monomeric T7 RNA polymerase,         via the flexible (Gly₃Ser)₄ linker. Each fusion proteins are         coexpressed with the other subunit of the vaccinia virus mRNA         capping enzyme, in order to form the heterodimeric         D12L/D1R-T7RNAP and D1R/D12L-T7RNAP chimeric enzymes,     -   two heterotrimeric chimeric enzymes, which are generated by         fusion of the EE₁₂₃₄L and RR₁₂₃₄L leucine-zippers to the         amino-terminal ends of one of the subunits of the vaccinia virus         mRNA capping enzyme and the T7 RNA polymerase, respectively.         Co-expression of RR₁₂₃₄L-D1R or pRR₁₂₃₄L-D12L, with the other         subunit of the vaccinia virus mRNA capping enzyme, plus         EE₁₂₃₄L-T7RNAP, form the heterotrimeric         D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP and         D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP chimeric enzymes.

I. Methods

The sequences used to generate the chimeric enzymes were assembled from synthetic oligonucleotides using a PCR-based method, cloned and fully sequence verified. These sequences were subcloned in the pCMV-Script plasmid with the subcloning cassette previously described. Finally, all the plasmids used for expression had the similar design: CMV IE1 promoter/enhancer promoter, Kozak sequence followed by the open-reading frames (ORFs), poly[A]-track, TΦ terminator for phage RNA polymerase transcription, and SV40 polyadenylation signal (FIG. 10 (A-H)).

As a consequence of the subcloning strategy, two amino-acids were added immediately downstream to the ATG of the Kozak sequence of some plasmids (Leu-Glu for pT7RNAP; Glu-Phe for pNP868R, pD1R, pD12L, pD1R-T7RNAP, and pD12L-T7RNAP), immediately downstream to the leucine-zipper sequences (Leu-Glu for pEE₁₂₃₄L-T7RNAP; Glu-Phe for pRR₁₂₃₄L-NP868R, pRR₁₂₃₄L-D1R and pRR₁₂₃₄L-D12L), and at the carboxyl-terminal end of some encoded proteins (Gly-Pro for pNP868R, pRR₁₂₃₄L-NP868R, pD1R, pD12L, pRR₁₂₃₄L-D1R and pRR₁₂₃₄L-D12L). In addition, two amino-acids were added immediately upstream (Gly-Pro for pD1R-T7RNAP and pD12L-T7RNAP) or downstream to the (Gly₃Ser)₄ linker (Leu-Glu for pD1R-T7RNAP and pD12L-T7RNAP).

As previously described, the Human Embryonic Kidney 293 cells (HEK-293) were grown in 24-wells plates. HEK-293 cells were transfected using the lipofectamine 2000 reagent, and the appropriate plasmids (0.4 μg DNA/well, plus 1 μL/well lipofectamine 2000, per transfected plasmid) as previously described. The firefly luciferase luminescence was assayed as previously described using the pET-22b(+)T7RNAPp-Luciferase reporter plasmid (designated pT7p-Luciferase thereafter) and the Luciferase Assay System. The expression of pORF-eSEAP plasmid was used to normalize the transfection efficacy as previously described.

Gene reporter expression was expressed as the luciferase luminescence in studied condition subtracted by the luminescence in cells treated with the transfection reagent only (RLU, relative light units), then divided by SEAP absorbance (OD, optic density) ratio. Statistical analyses were performed using Student's t two-tailed test adjusted by Holm-Bonferroni correction for multiple testing, if appropriate. A p-value of less than 0.05 was regarded as being statistically significant.

II. Results

II.1 Heterodimeric RR₁₂₃₄L-NP868R/EE₁₂₃₄L-T7RNAP Chimeric Enzyme

The activity of the heterodimeric enzyme RR₁₂₃₄L-NP868R/EE₁₂₃₄L-T7RNA chimeric enzyme (encoded by pRR₁₂₃₄L-pNP868R and pEE₁₂₃₄L-T7RNAP plasmids, respectively) has been demonstrated. This heterodimeric enzyme is generated by non-covalent linkage between the monomeric African Swine Fever Virus mRNA capping enzyme pNP868R and the monomeric T7 RNA polymerase, via the EE₁₂₃₄L and RR₁₂₃₄L leucine-zippers (FIG. 9). The EE₁₂₃₄L (acidic leucine-zipper; LEIAAFLEQENTALETEVAELEQEVQRLENIVSQYETRYGPLGGGK (SEQ ID NO:51), one letter amino-acid code) and RR₁₂₃₄L leucine-zippers (basic leucine-zipper with slight modification of the GGGK orientation, which is not involved in leucine-zipper dimerization (Moll, Ruvinov et al. 2001); LEIRAAFLRRRNTALRTRVAELRQRVQRLRNIVSQYETRYGPLGGGK (SEQ ID NO:52), one letter amino-acid code), which were respectively added to the amino-terminal end of NP868R and T7RNAP, respectively. The RR₁₂₃₄L and EE₁₂₃₄L leucine zippers are dimeric coiled-coil peptide structures consisting of two amphipathic α-helices that preferably melt as heterodimer in antiparallel orientation (Moll, Ruvinov et al. 2001).

As expected, the transfection of the plasmid encoding for the African Swine Fever Virus mRNA capping enzyme alone with or without leucine-zipper sequences (i.e. pRR₁₂₃₄L-NP868R and pNP868R, respectively) do not induce any detectable luciferase reporter gene expression (FIG. 11). As also expected, cells transfected with the plasmid encoding for the T7 RNA polymerase with the EE₁₂₃₄L leucine zipper (encoded by pEE₁₂₃₄L-T7RNA) show very similar activity to the T7 RNA polymerase without leucine-zipper (encoded by pT7RNA), which provide further evidence that the native amino-terminal end of the T7 RNA polymerase can be modified without major impairment of its enzymatic processivity.

The HEK293 cells co-transfected with the pRR₁₂₃₄L-NP868R and pEE₁₂₃₄L-T7RNA plasmids (encoding for NP868R and T7RNAP with leucine-zippers), together with the reporter pT7p-luciferase plasmid, show strong luciferase reporter gene expression signal, which is 87% to that of pCMV-T7RNAP plasmid (non-statistically significant difference, Student's t-test; (FIG. 11). Furthermore, cells co-transfected in presence of the reporter pT7p-luciferase plasmid with the pRR₁₂₃₄L-NP868R and pEE₁₂₃₄L-T7RNA plasmids, displays 3.7-fold higher luciferase reporter gene expression than cells cotransfected with pNP868R and pT7RNAP (encoding for NP868R and T7RNAP without leucine-zippers; p<0.05, Student's t-test).

These results demonstrate the activity of heterodimeric chimeric enzymes according to the invention and that the non-covalent linkage between NP868R and T7RNAP by leucine-zippers increases significantly the expression of the gene reporter driven by said chimeric enzymes.

II.2 Heterodimeric D1R/D12L-T7RNAP and D12L/D1R-T7RNAP Chimeric Enzymes

The activity of other types of heterodimeric chimeric enzyme has also been demonstrated, using the vaccinia mRNA capping enzyme.

By itself, the vaccinia mRNA capping enzyme is a heterodimer consisting of: (i) a 95 kDa subunit encoded by the vaccinia virus D1R gene (genomic sequence ID# NC_006998.1; GeneID#3707562; UniProtKB/Swiss-Prot ID# YP_232988.1), designated hereafter as D1R, which has RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine methyltransferase enzymatic activities (Cong and Shuman 1993; Niles and Christen 1993; Higman and Niles 1994; Mao and Shuman 1994; Gong and Shuman 2003), (ii) and a 31-kDa subunit encoded by the vaccinia virus D12L gene (genomic sequence ID# NC_006998.1; GeneID#3707515; UniProtKB/Swiss-Prot ID#YP_232999.1), designated hereafter as D12L, which has no intrinsic enzymatic activity, but enhances drastically the RNA N7-guanine methyltransferase activity of the D1R subunit (Higman, Bourgeois et al. 1992; Higman, Christen et al. 1994; Mao and Shuman 1994).

D1R or D12L were fused to the amino-terminal end of the T7 RNA polymerase, via the (Gly₃Ser)₄ linker (encoded by D1R-T7RNAP or D12L-T7RNAP, respectively). When co-expressed, each fusion proteins, together with the other vaccinia mRNA capping enzyme subunit (encoded by pD12L or D1R, respectively), generate two different heterodimeric chimeric enzymes designated as D12L/D1R-T7RNAP and D1R/D12L-T7RNAP, respectively (FIG. 9). In presence of the reporter pT7p-luciferase plasmid, the heterodimeric chimeric enzymes generate a strong luciferase gene reporter signal in HEK293 cells (FIG. 12). The heterodimeric D12L/D1R-T7RNAP chimeric enzyme has 32% activity to that of pCMV-T7RNAP plasmid (p<0.01, Student's t-test), whereas the D1R/D12L-T7RNAP heterodimeric enzyme has 1.5-fold higher luciferase reporter gene expression than the pCMV-T7RNAP plasmid (non-statistically significant difference, Student's t-test). Furthermore, the coexpression of the two subunits of vaccinia mRNA capping enzyme unbound to the T7 RNA polymerase (encoded by pD1R, pD12L and pT7RNAP) shows 9- and 42-fold lower luciferase reporter gene expression signal than the heterodimeric D12L/D1R-T7RNAP and D1R/D12L-T7RNAP chimeric enzymes, respectively (P<0.05 for both statistical comparison, Student's t-test).

These results demonstrate the activity of different types of heterodimeric chimeric enzymes according to the invention and that covalent linkage between the subunits of the vaccinia mRNA capping enzyme and the T7RNAP stimulates significantly the gene reporter expression. As also expected, in presence of the reporter pT7p-luciferase plasmid, the expression of D1R and/or D12L without T7 RNA polymerase induces virtually no detectable luciferase expression.

II.3 Heterotrimeric D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP and D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP Chimeric Enzymes

The activity of heterotrimeric chimeric enzyme has also been demonstrated.

The basic RR₁₂₃₄L leucine zipper was fused to the amino-terminal ends of either the D1R or D12L subunits of the vaccinia virus mRNA capping enzyme (encoded by pRR₁₂₃₄L-D1R and RR₁₂₃₄L-D12L, respectively), while the complementary acidic EE₁₂₃₄L leucine-zipper was added to the amino-terminal end of T7 RNA polymerase (encoded by the pEE₁₂₃₄L-T7RNA plasmid). The co-expression of pEE₁₂₃₄L-T7RNAP, together with either pRR₁₂₃₄L-D1R or pRR₁₂₃₄L-D12L, plus the other vaccinia mRNA capping enzyme subunit (pD12L and pD1R plasmids, respectively), therefore generate two different heterotrimeric CCPP enzymes, designated as D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP and D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP, respectively (FIG. 9).

The T7 RNA polymerase displayed 7-fold higher luciferase gene reporter signal when coexpressed with the D1R/D12L subunits of the vaccinia virus mRNA capping enzyme than in their absence. These results are therefore in line with those obtained by the vaccinia virus/bacteriophage RNAP hybrid expression system, in which the translatability of uncapped T7 transcripts is increased by the expression of the vaccinia mRNA capping enzyme provided by a recombinant virus (Fuerst, Niles et al. 1986; Fuerst, Earl et al. 1987; Elroy-Stein, Fuerst et al. 1989; Fuerst, Fernandez et al. 1989; Fuerst and Moss 1989; Elroy-Stein and Moss 1990).

A strong luciferase gene reporter signal was shown in HEK-293 cells expressing either the D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP or the D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP CCPP enzymes, in presence of the reporter pT7p-luciferase plasmid (FIG. 13). The heterodimeric D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP and D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP chimeric enzymes have respectively 57% and 33% activity to that of pCMV-T7RNAP plasmid (non-statistically significant difference, Student's t-test). The heterodimeric D12L/RR₁₂₃₄L-D1R/EE₁₂₃₄L-T7RNAP and D1R/RR₁₂₃₄L-D12L/EE₁₂₃₄L-T7RNAP chimeric enzymes show respectively 11- and 6.7-fold stronger luciferase gene reporter expression signal than cells expressing D1, D12L and T7RNAP without leucine-zippers (p=0.05 and non-statistically significant difference, respectively; Student's t-test).

These results demonstrate the activity of heterotrimeric chimeric enzymes according to the invention and that the non-covalent linkage between any of the subunits of the vaccinia mRNA capping enzyme and the T7 RNA polymerase increases significantly the gene reporter expression.

III. Conclusion

These present results show the activity of different types of heterodimeric and heterotrimeric chimeric enzymes according to the invention, generated by covalent or non-covalent linkage.

The present results also provide evidences that covalent or non-covalent linkage between the different catalytic domain of the chimeric enzyme and in particular between capping enzymes and RNA polymerases allows the optimization of the gene reporter expression by the chimeric enzymes.

Example 4 Stimulation of Luciferase Reporter Gene Expression by Silencing Sequences Against the Cellular RNA Polymerase II and Capping Enzyme

I. Methods

HEK-293 cells were grown as previously described in 24-wells plates and transfected using the Lipofectamine 2000 reagent, and the appropriate concentration of siRNA (Qiagen; Hilden, Germany) and plasmids (0.4 μg DNA/well, plus 1 μL/well lipofectamine 2000, per transfected plasmid). The NP868R-SP6 chimeric enzyme, which has strong demonstrated activity, was used in the present experiment. The firefly luciferase luminescence was assayed as previously described using the pT7p-Luciferase (which also contains both the T3 and SP6 promoters) and the Luciferase Assay System. The expression of pORF-eSEAP plasmid was used to normalize for transfection efficacy as previously described.

Four siRNA that target the human POLR2A (NCBI Gene ID#5430; mRNA sequence ID# NM_000937.4; NCBI protein sequence ID# NP_000928.1) were used: SI04364381 (mRNA sequence 1255-1275: CAGCGGTTGAAGGGCAAGGAA (SEQ ID NO:43)), SI04369344 (mRNA sequence 830-850: ATGCGGAATGGAAGCACGTTA (SEQ ID NO:44)), SI04250162 (mRNA sequence 2539-2559: ATGGTCGTGTCCGGAGCTAAA (SEQ ID NO:45)), and SI04354420 (mRNA sequence 4896-4916: CAGCGGCTTCAGCCCAGGTTA (SEQ ID NO:46)).

In addition, four siRNA that target the human RNGTT (Gene ID#8732; mRNA sequence ID# NM_003800.3; NCBI protein sequence ID# NP_003791.3) were used: SI00055986 (mRNA sequence 3187-3207: ATGGATTTAAAGGGCGGCTAA (SEQ ID NO:47)), SI03021508 (mRNA sequence 430-450: TTCAAGGTTCTATGACCGAAA (SEQ ID NO:48)), SI00055972 (mRNA sequence 2530-2550: CAGGGTTGTTAAGTTGTACTA (SEQ ID NO:49)) and SI00055979 (mRNA sequence 4132-4152: TACCATCTGCAGTATTATAAA (SEQ ID NO:50)).

II. Results

In a first series of experiments, the effects of four POLR2A siRNA and four RNGTT siRNA were tested at 25 nM final concentration (FIG. 14). The siRNA were co-transfected with the pNP868R-SP6RNAP chimeric enzyme plasmid and the reporter pT7p-luciferase plasmid. Collectively, the POLR2A siRNA trend to increase the luciferase gene reporter expression by 127% on average, in comparison the same condition without siRNA. Similarly, the addition of RNGTT siRNA collectively increased the luciferase gene reporter expression to 147% on average in comparison the same condition without siRNA.

The POLR2A SI04369344 and the RNGTT SI00055972 siRNA, which have show the highest stimulation rate, were selected for a second series of experiments. Expression of the luciferase reporter gene driven by NP868R-SP6RNAP was assayed in presence of siRNA at concentrations ranging from 0 to 100 nM (FIG. 15). Dose-response was observed with both siRNA. The strongest expression stimulation of 3.8-folds was observed at 100 nM with POLR2A SI04369344, and of 5.1-folds with the RNGTT SI00055972 siRNA at 100 nM.

III. Conclusion

The present findings demonstrate that the silencing of the cellular transcription and post-transcriptional machineries by siRNA stimulate the reporter gene expression driven by the NP868R-SP6RNAP chimeric enzyme.

BIBLIOGRAPHIC REFERENCES

-   Bedzyk, W. D., K. M. Weidner, et al. (1990). “Immunological and     structural characterization of a high affinity anti-fluorescein     single-chain antibody.” J Biol Chem 265(30): 18615-18620. -   Bengal, E., 0. Flores, et al. (1991). “Role of the mammalian     transcription factors IIF, IIS, and IIX during elongation by RNA     polymerase II.” Mol Cell Biol 11(3): 1195-1206. -   Benton, B. M., W. K. Eng, et al. (1990). “Signal-mediated import of     bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae     nucleus and specific transcription of target genes.” Mol Cell Biol     10(1): 353-360. -   Bird, R. E., K. D. Hardman, et al. (1988). “Single-chain     antigen-binding proteins.” Science 242(4877): 423-426. -   Bridgen, A. and R. M. Elliott (1996). “Rescue of a segmented     negative-strand RNA virus entirely from cloned complementary DNAs.”     Proc Natl Acad Sci USA 93(26): 15400-15404. -   Brisson, M., Y. He, et al. (1999). “A novel T7 RNA polymerase     autogene for efficient cytoplasmic expression of target genes.” Gene     Ther 6(2): 263-270. -   Busch, R., A. Pashine, et al. (2002). “Stabilization of soluble,     low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers.” J Immunol     Methods 263(1-2): 111-121. -   Busch, R., Z. Reich, et al. (1998). “Secondary structure composition     and pH-dependent conformational changes of soluble recombinant     HLA-DM.” J Biol Chem 273(42): 27557-27564. -   Bushnell, D. A., P. Cramer, et al. (2002). “Structural basis of     transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A     resolution.” Proc Natl Acad Sci USA 99(3): 1218-1222. -   Chang, H. C., Z. Bao, et al. (1994). “A general method for     facilitating heterodimeric pairing between two proteins: application     to expression of alpha and beta T-cell receptor extracellular     segments.” Proc Natl Acad Sci USA 91(24): 11408-11412. -   Chen, X., Y. Li, et al. (1994). “A self-initiating eukaryotic     transient gene expression system based on contransfection of     bacteriophage T7 RNA polymerase and DNA vectors containing a T7     autogene.” Nucleic Acids Res 22(11): 2114-2120. -   Chen, Z. and T. D. Schneider (2005). “Information theory based     T7-like promoter models: classification of bacteriophages and     differential evolution of promoters and their polymerases.” Nucleic     Acids Res 33(19): 6172-6187. -   Cho, E. J., T. Takagi, et al. (1997). “mRNA capping enzyme is     recruited to the transcription complex by phosphorylation of the RNA     polymerase II carboxy-terminal domain.” Genes Dev 11(24): 3319-3326. -   Clayton, D. A. (1991). “Replication and transcription of vertebrate     mitochondrial DNA.” Annu Rev Cell Biol 7: 453-478. -   Cong, P. and S. Shuman (1993). “Covalent catalysis in nucleotidyl     transfer. A KTDG motif essential for enzyme-GMP complex formation by     mRNA capping enzyme is conserved at the active sites of RNA and DNA     ligases.” J Biol Chem 268(10): 7256-60. -   Cong, Y. S., D. Yarrow, et al. (1994). “Linear DNA plasmids from     Pichia etchellsii, Debaryomyces hansenii and Wingea robertsiae.”     Microbiology 140 (Pt 6): 1327-1335. -   Conzelmann, K. K. and M. Schnell (1994). “Rescue of synthetic     genomic RNA analogs of rabies virus by plasmid-encoded proteins.” J     Virol 68(2): 713-719. -   Crasto, C. J. and J. A. Feng (2000). “LINKER: a program to generate     linker sequences for fusion proteins.” Protein Eng 13(5): 309-312. -   Cronan, J. E., Jr. (1990). “Biotination of proteins in vivo. A     post-translational modification to label, purify, and study     proteins.” J Biol Chem 265(18): 10327-33. -   Davanloo, P., A. H. Rosenberg, et al. (1984). “Cloning and     expression of the gene for bacteriophage T7 RNA polymerase.” Proc     Natl Acad Sci USA 81(7): 2035-2039. -   Dias, N. and C. A. Stein (2002). “Antisense oligonucleotides: basic     concepts and mechanisms.” Mol Cancer Ther 1(5): 347-55. -   Dower, K. and M. Rosbash (2002). “T7 RNA polymerase-directed     transcripts are processed in yeast and link 3′ end formation to mRNA     nuclear export.” Rna 8(5): 686-697. -   Drummond, D. R., J. Armstrong, et al. (1985). “The effect of capping     and polyadenylation on the stability, movement and translation of     synthetic messenger RNAs in Xenopus oocytes.” Nucleic Acids Res     13(20): 7375-7394. -   Elroy-Stein, O., T. R. Fuerst, et al. (1989). “Cap-independent     translation of mRNA conferred by encephalomyocarditis virus 5′     sequence improves the performance of the vaccinia     virus/bacteriophage T7 hybrid expression system.” Proc Natl Acad Sci     USA 86(16): 6126-6130. -   Elroy-Stein, O. and B. Moss (1990). “Cytoplasmic expression system     based on constitutive synthesis of bacteriophage T7 RNA polymerase     in mammalian cells.” Proc Natl Acad Sci USA 87(17): 6743-6747. -   Engleka, K. A., E. W. Lewis, et al. (1998). “Mechanisms of     replication-deficient vaccinia virus/T7 RNA polymerase hybrid     expression: effect of T7 RNA polymerase levels and alpha-amanitin.”     Virology 243(2): 331-339. -   Epicentre Biotechnologies website. “ScriptCap™     2′-O-Methyltransferase.” -   Fancy, D. A. and T. Kodadek (1999). “Chemistry for the analysis of     protein-protein interactions: rapid and efficient cross-linking     triggered by long wavelength light.” Proc Natl Acad Sci USA 96(11):     6020-4. -   Fernandez-Silva, P., F. Martinez-Azorin, et al. (1997). “The human     mitochondrial transcription termination factor (mTERF) is a     multizipper protein but binds to DNA as a monomer, with evidence     pointing to intramolecular leucine zipper interactions.” Embo J     16(5): 1066-1079. -   Finn, J., I. MacLachlan, et al. (2005). “Factors limiting     autogene-based cytoplasmic expression systems.” Faseb J 19(6):     608-610. -   Fire, A., M. Samuels, et al. (1984). “Interactions between RNA     polymerase II, factors, and template leading to accurate     transcription.” J Biol Chem 259(4): 2509-2516. -   Fisher, R. P. and D. A. Clayton (1985). “A transcription factor     required for promoter recognition by human mitochondrial RNA     polymerase. Accurate initiation at the heavy- and light-strand     promoters dissected and reconstituted in vitro.” J Biol Chem     260(20): 11330-11338. -   Fisher, R. P. and D. A. Clayton (1988). “Purification and     characterization of human mitochondrial transcription factor 1.” Mol     Cell Biol 8(8): 3496-3509. -   Fisher, R. P., J. N. Topper, et al. (1987). “Promoter selection in     human mitochondria involves binding of a transcription factor to     orientation-independent upstream regulatory elements.” Cell 50(2):     247-258. -   Fortes, P., Y. Cuevas, et al. (2003). “Inhibiting expression of     specific genes in mammalian cells with 5′ end-mutated U1 small     nuclear RNAs targeted to terminal exons of pre-mRNA.” Proc Natl Acad     Sci USA 100(14): 8264-9. -   Fuerst, T. R., P. L. Earl, et al. (1987). “Use of a hybrid vaccinia     virus-T7 RNA polymerase system for expression of target genes.” Mol     Cell Biol 7(7): 2538-2544. -   Fuerst, T. R., M. P. Fernandez, et al. (1989). “Transfer of the     inducible lac repressor/operator system from Escherichia coli to a     vaccinia virus expression vector.” Proc Natl Acad Sci USA 86(8):     2549-2553. -   Fuerst, T. R. and B. Moss (1989). “Structure and stability of mRNA     synthesized by vaccinia virus-encoded bacteriophage T7 RNA     polymerase in mammalian cells. Importance of the 5′ untranslated     leader.” J Mol Biol 206(2): 333-348. -   Fuerst, T. R., E. G. Niles, et al. (1986). “Eukaryotic     transient-expression system based on recombinant vaccinia virus that     synthesizes bacteriophage T7 RNA polymerase.” Proc Natl Acad Sci USA     83(21): 8122-8126. -   Furuichi, Y., A. LaFiandra, et al. (1977). “5′-Terminal structure     and mRNA stability.” Nature 266(5599): 235-239. -   Furuichi, Y. and A. J. Shatkin (2000). “Viral and cellular mRNA     capping: past and prospects.” Adv Virus Res 55: 135-184. -   Gallie, D. R. (1991). “The cap and poly(A) tail function     synergistically to regulate mRNA translational efficiency.” Genes     Dev 5(11): 2108-2116. -   Gao, X. and L. Huang (1993). “Cytoplasmic expression of a reporter     gene by co-delivery of T7 RNA polymerase and T7 promoter sequence     with cationic liposomes.” Nucleic Acids Res 21(12): 2867-2872. -   Garcin, D., T. Pelet, et al. (1995). “A highly recombinogenic system     for the recovery of infectious Sendai paramyxovirus from cDNA:     generation of a novel copy-back nondefective interfering virus.”     Embo J 14(24): 6087-6094. -   Ghosh, I., A. D. Hamilton, et al. (2000). “Antiparallel Leucine     Zipper-Directed Protein Reassembly:&nbsp; Application to the Green     Fluorescent Protein.” Journal of the American Chemical Society     122(23): 5658-5659. -   Gilbert, W. and A. Maxam (1973). “The nucleotide sequence of the lac     operator.” Proc Natl Acad Sci USA 70(12): 3581-3584. -   Gill, D. R., S. E. Smyth, et al. (2001). “Increased persistence of     lung gene expression using plasmids containing the ubiquitin C or     elongation factor 1alpha promoter.” Gene Ther 8(20): 1539-1546. -   Gingras, A. C., B. Raught, et al. (1999). “eIF4 initiation factors:     effectors of mRNA recruitment to ribosomes and regulators of     translation.” Annu Rev Biochem 68: 913-963. -   Golomb, M. and M. Chamberlin (1974). “Characterization of     T7-specific ribonucleic acid polymerase. IV. Resolution of the major     in vitro transcripts by gel electrophoresis.” J Biol Chem 249(9):     2858-2863. -   Gong, C. and S. Shuman (2003). “Mapping the active site of vaccinia     virus RNA triphosphatase.” Virology 309(1): 125-34. -   Gregoire, C., S. Y. Lin, et al. (1996). “Covalent assembly of a     soluble T cell receptor-peptide-major histocompatibility class I     complex.” Proc Natl Acad Sci USA 93(14): 7184-7189. -   Gustavsson, M., J. Lehtio, et al. (2001). “Stable linker peptides     for a cellulose-binding domain-lipase fusion protein expressed in     Pichia pastoris.” Protein Eng 14(9): 711-715. -   Han, Y. T., C. S. Tsai, et al. (2007). “Mutational analysis of a     helicase motif-based RNA 5′-triphosphatase/NTPase from bamboo mosaic     virus.” Virology. -   Hennecke, F., C. Krebber, et al. (1998). “Non-repetitive     single-chain Fv linkers selected by selectively infective phage     (SIP) technology.” Protein Eng 11(5): 405-410. -   Higman, M. A., N. Bourgeois, et al. (1992). “The vaccinia virus mRNA     (guanine-N7-)-methyltransferase requires both subunits of the mRNA     capping enzyme for activity.” J Biol Chem 267(23): 16430-7. -   Higman, M. A., L. A. Christen, et al. (1994). “The mRNA     (guanine-7-)methyltransferase domain of the vaccinia virus mRNA     capping enzyme. Expression in Escherichia coli and structural and     kinetic comparison to the intact capping enzyme.” J Biol Chem     269(21): 14974-81. -   Higman, M. A. and E. G. Niles (1994). “Location of the     S-adenosyl-L-methionine binding region of the vaccinia virus mRNA     (guanine-7-)methyltransferase.” J Biol Chem 269(21): 14982-7. -   Hu, W., F. Li, et al. (2004). “A flexible peptide linker enhances     the immunoreactivity of two copies HBsAg preS1 (21-47) fusion     protein.” J Biotechnol 107(1): 83-90. -   Huang, Y. and J. A. Steitz (2005). “SRprises along a messenger's     journey.” Mol Cell 17(5): 613-615. -   Huston, J. S., D. Levinson, et al. (1988). “Protein engineering of     antibody binding sites: recovery of specific activity in an     anti-digoxin single-chain Fv analogue produced in Escherichia coli.”     Proc Natl Acad Sci USA 85(16): 5879-5883. -   Izban, M. G. and D. S. Luse (1992). “Factor-stimulated RNA     polymerase II transcribes at physiological elongation rates on naked     DNA but very poorly on chromatin templates.” J Biol Chem 267(19):     13647-13655. -   Jacob, S. T., E. M. Sajdel, et al. (1970). “Specific action of     alpha-amanitin on mammalian RNA polymerase protein.” Nature     225(5227): 60-62. -   Kedinger, C., M. Gniazdowski, et al. (1970). “Alpha-amanitin: a     specific inhibitor of one of two DNA-pendent RNA polymerase     activities from calf thymus.” Biochem Biophys Res Commun 38(1):     165-171. -   Kohler, A. and E. Hurt (2007). “Exporting RNA from the nucleus to     the cytoplasm.” Nat Rev Mol Cell Biol 8(10): 761-773. -   Komarnitsky, P., E. J. Cho, et al. (2000). “Different phosphorylated     forms of RNA polymerase II and associated mRNA processing factors     during transcription.” Genes Dev 14(19): 2452-2460. -   Kozak, M. (1987). “At least six nucleotides preceding the AUG     initiator codon enhance translation in mammalian cells.” J Mol Biol     196(4): 947-950. -   Kozak, M. (2005). “Regulation of translation via mRNA structure in     prokaryotes and eukaryotes.” Gene 361: 13-37. -   Kruse, B., N. Narasimhan, et al. (1989). “Termination of     transcription in human mitochondria: identification and purification     of a DNA binding protein factor that promotes termination.” Cell     58(2): 391-397. -   Kuge, H., G. G. Brownlee, et al. (1998). “Cap ribose methylation of     c-mos mRNA stimulates translation and oocyte maturation in Xenopus     laevis.” Nucleic Acids Res 26(13): 3208-3214. -   Kupper, H. A., W. T. McAllister, et al. (1973). “Comparison of     Escherichia coli and T3 RNA polymerases. Differential inhibition of     transcription by various drugs.” Eur J Biochem 38(3): 581-586. -   Lamla, T. and V. A. Erdmann (2004). “The Nano-tag, a     streptavidin-binding peptide for the purification and detection of     recombinant proteins.” Protein Expr Purif 33(1): 39-47. -   Lang, I., M. Scholz, et al. (1986). “Molecular mobility and     nucleocytoplasmic flux in hepatoma cells.” J Cell Biol 102(4):     1183-1190. -   Langberg, S. R. and B. Moss (1981). “Post-transcriptional     modifications of mRNA. Purification and characterization of cap I     and cap II RNA (nucleoside-2′-)-methyltransferases from HeLa cells.”     J Biol Chem 256(19): 10054-10060. -   Lee, D. N., L. Phung, et al. (1990). “Transcription pausing by     Escherichia coli RNA polymerase is modulated by downstream DNA     sequences.” J Biol Chem 265(25): 15145-15153. -   Li, Y. I., Y. J. Chen, et al. (2001). “Characterization of the     AdoMet-dependent guanylyltransferase activity that is associated     with the N terminus of bamboo mosaic virus replicase.” J Virol     75(2): 782-788. -   Li, Y. I., Y. M. Cheng, et al. (1998). “Identification and     characterization of the Escherichia coli-expressed RNA-dependent RNA     polymerase of bamboo mosaic virus.” J Virol 72(12): 10093-10099. -   Li, Y. I., T. W. Shih, et al. (2001). “The helicase-like domain of     plant potexvirus replicase participates in formation of RNA 5′ cap     structure by exhibiting RNA 5′-triphosphatase activity.” J Virol     75(24): 12114-12120. -   Lian, Y., M. B. De Young, et al. (1999). “The sCYMV1 hairpin     ribozyme: targeting rules and cleavage of heterologous RNA.” Gene     Ther 6(6): 1114-9. -   Lieschke, G. J., P. K. Rao, et al. (1997). “Bioactive murine and     human interleukin-12 fusion proteins which retain antitumor activity     in vivo.” Nat Biotechnol 15(1): 35-40. -   Lindell, T. J., F. Weinberg, et al. (1970). “Specific inhibition of     nuclear RNA polymerase II by alpha-amanitin.” Science 170(956):     447-449. -   Lisser, S. and H. Margalit (1993). “Compilation of E. coli mRNA     promoter sequences.” Nucleic Acids Res 21(7): 1507-1516. -   Liu, Z. and G. G. Carmichael (1994). “Nuclear antisense RNA. An     efficient new method to inhibit gene expression.” Mol Biotechnol     2(2): 107-18. -   Liu, Z., J. Jian-Bo, et al. (2005). “Anti-proteolysis Study of     Recombinant IIn-UK Fusion Protein in CHO Cell” Prog. Biochem.     Biophys 32(6): 544-550. -   Lo, H. J., H. K. Huang, et al. (1998). “RNA polymerase I-promoted     HIS4 expression yields uncapped, polyadenylated mRNA that is     unstable and inefficiently translated in Saccharomyces cerevisiae.”     Mol Cell Biol 18(2): 665-675. -   Lodish, H., A. Berk, et al. (2008). Molecular Cell Biology. New     York, USA, Freeman, W. H. and Co. -   Loser, P., G. S. Jennings, et al. (1998). “Reactivation of the     previously silenced cytomegalovirus major immediate-early promoter     in the mouse liver: involvement of NFkappaB.” J Virol 72(1):     180-190. -   Lumb, K. J. and P. S. Kim (1995). “A buried polar interaction     imparts structural uniqueness in a designed heterodimeric coiled     coil.” Biochemistry 34(27): 8642-8. -   Lyakhov, D. L., B. He, et al. (1997). “Mutant bacteriophage T7 RNA     polymerases with altered termination properties.” J Mol Biol 269(1):     28-40. -   Makarova, O. V., E. M. Makarov, et al. (1995). “Transcribing of     Escherichia coli genes with mutant T7 RNA polymerases: stability of     lacZ mRNA inversely correlates with polymerase speed.” Proc Natl     Acad Sci USA 92(26): 12250-12254. -   Malone, R. W., P. L. Felgner, et al. (1989). “Cationic     liposome-mediated RNA transfection.” Proc Natl Acad Sci USA 86(16):     6077-6081. -   Mantile, G., C. Fuchs, et al. (2000). “Stable, long-term bacterial     production of soluble, dimeric, disulfide-bonded protein     pharmaceuticals without antibiotic selection.” Biotechnol Prog     16(1): 17-25. -   Mao, X. and S. Shuman (1994). “Intrinsic RNA (guanine-7)     methyltransferase activity of the vaccinia virus capping enzyme D1     subunit is stimulated by the D12 subunit. Identification of amino     acid residues in the D1 protein required for subunit association and     methyl group transfer.” J Biol Chem 269(39): 24472-9. -   Martinez-Costas, J., G. Sutton, et al. (1998). “Guanylyltransferase     and RNA 5′-triphosphatase activities of the purified expressed VP4     protein of bluetongue virus.” J Mol Biol 280(5): 859-866. -   McClain, D. L., H. L. Woods, et al. (2001). “Design and     characterization of a heterodimeric coiled coil that forms     exclusively with an antiparallel relative helix orientation.” J Am     Chem Soc 123(13): 3151-3152. -   McCracken, S., N. Fong, et al. (1997). “5′-Capping enzymes are     targeted to pre-mRNA by binding to the phosphorylated     carboxy-terminal domain of RNA polymerase II.” Genes Dev 11(24):     3306-3318. -   McCulloch, V., B. L. Seidel-Rogol, et al. (2002). “A human     mitochondrial transcription factor is related to RNA adenine     methyltransferases and binds S-adenosylmethionine.” Mol Cell Biol     22(4): 1116-1125. -   Miao, C. H., A. R. Thompson, et al. (2001). “Long-term and     therapeutic-level hepatic gene expression of human factor IX after     naked plasmid transfer in vivo.” Mol Ther 3(6): 947-957. -   Miao, C. H., X. Ye, et al. (2003). “High-level factor VIII gene     expression in vivo achieved by nonviral liver-specific gene therapy     vectors.” Hum Gene Ther 14(14): 1297-1305. -   Mifflin, R. C. and R. E. Kellems (1991). “Coupled     transcription-polyadenylation in a cell-free system.” J Biol Chem     266(29): 19593-19598. -   Moffatt, B. A., J. J. Dunn, et al. (1984). “Nucleotide sequence of     the gene for bacteriophage T7 RNA polymerase.” J Mol Biol 173(2):     265-269. -   Moll, J. R., S. B. Ruvinov, et al. (2001). “Designed     heterodimerizing leucine zippers with a ranger of pls and     stabilities up to 10(−15) M.” Protein Sci 10(3): 649-55. -   Natalizio, B. J., N. D. Robson-Dixon, et al. (2009). “The     Carboxyl-terminal Domain of RNA Polymerase II Is Not Sufficient to     Enhance the Efficiency of Pre-mRNA Capping or Splicing in the     Context of a Different Polymerase.” J Biol Chem 284(13): 8692-8702. -   Newton, D. L., Y. Xue, et al. (1996). “Angiogenin single-chain     immunofusions: influence of peptide linkers and spacers between     fusion protein domains.” Biochemistry 35(2): 545-553. -   Nicol, F., M. Wong, et al. (2002). “Poly-L-glutamate, an anionic     polymer, enhances transgene expression for plasmids delivered by     intramuscular injection with in vivo electroporation.” Gene Ther     9(20): 1351-1358. -   Niles, E. G. and L. Christen (1993). “Identification of the vaccinia     virus mRNA guanyltransferase active site lysine.” J Biol Chem     268(33): 24986-9. -   O'Shea, E. K., J. D. Klemm, et al. (1991). “X-ray structure of the     GCN4 leucine zipper, a two-stranded, parallel coiled coil.” Science     254(5031): 539-544. -   O'Shea, E. K., K. J. Lumb, et al. (1993). “Peptide ‘Velcro’: design     of a heterodimeric coiled coil.” Curr Biol 3(10): 658-667. -   Oakley, M. G. and P. S. Kim (1998). “A buried polar interaction can     direct the relative orientation of helices in a coiled coil.”     Biochemistry 37(36): 12603-12610. -   Ojala, D., J. Montoya, et al. (1981). “tRNA punctuation model of RNA     processing in human mitochondria.” Nature 290(5806): 470-474. -   Osumi-Davis, P. A., M. C. de Aguilera, et al. (1992). “Asp537,     Asp812 are essential and Lys631, His811 are catalytically     significant in bacteriophage T7 RNA polymerase activity.” J Mol Biol     226(1): 37-45. -   Osumi-Davis, P. A., N. Sreerama, et al. (1994). “Bacteriophage T7     RNA polymerase and its active-site mutants. Kinetic, spectroscopic     and calorimetric characterization.” J Mol Biol 237(1): 5-19. -   Paguirigan, A. L. and D. J. Beebe (2007). “Protocol for the     fabrication of enzymatically crosslinked gelatin microchannels for     microfluidic cell culture.” Nat Protoc 2(7): 1782-8. -   Palancade, B. and O. Bensaude (2003). “Investigating RNA polymerase     II carboxyl-terminal domain (CTD) phosphorylation.” Eur J Biochem     270(19): 3859-3870. -   Pantoliano, M. W., R. E. Bird, et al. (1991). “Conformational     stability, folding, and ligand-binding affinity of single-chain Fv     immunoglobulin fragments expressed in Escherichia coli.”     Biochemistry 30(42): 10117-10125. -   Pashine, A., R. Busch, et al. (2003). “Interaction of HLA-DR with an     acidic face of HLA-DM disrupts sequence-dependent interactions with     peptides.” Immunity 19(2): 183-192. -   Patil, S. D., D. G. Rhodes, et al. (2004). “Anionic liposomal     delivery system for DNA transfection.” Aaps J 6(4): e29. -   Pavlinkova, G., G. W. Beresford, et al. (1999). “Pharmacokinetics     and biodistribution of engineered single-chain antibody constructs     of MAb CC49 in colon carcinoma xenografts.” J Nucl Med 40(9):     1536-1546. -   Platt, T. (1986). “Transcription termination and the regulation of     gene expression.” Annu Rev Biochem 55: 339-372. -   Prieto-Martin, A., J. Montoya, et al. (2001). “A study on the human     mitochondrial RNA polymerase activity points to existence of a     transcription factor B-like protein.” FEBS Lett 503(1): 51-55. -   Ramadevi, N., N. J. Burroughs, et al. (1998). “Capping and     methylation of mRNA by purified recombinant VP4 protein of     bluetongue virus.” Proc Natl Acad Sci USA 95(23): 13537-13542. -   Ramadevi, N., J. Rodriguez, et al. (1998). “A leucine zipper-like     domain is essential for dimerization and encapsidation of bluetongue     virus nucleocapsid protein VP4.” J Virol 72(4): 2983-2990. -   Ramsey-Ewing, A. and B. Moss (1996). “Recombinant protein synthesis     in Chinese hamster ovary cells using a vaccinia virus/bacteriophage     T7 hybrid expression system.” J Biol Chem 271(28): 16962-16966. -   Rhoads, R. E. (1999). “Signal transduction pathways that regulate     eukaryotic protein synthesis.” J Biol Chem 274(43): 30337-30340. -   Robinson, C. R. and R. T. Sauer (1998). “Optimizing the stability of     single-chain proteins by linker length and composition mutagenesis.”     Proc Natl Acad Sci USA 95(11): 5929-5934. -   Salehi-Ashtiani, K. and J. W. Szostak (2001). “In vitro evolution     suggests multiple origins for the hammerhead ribozyme.” Nature     414(6859): 82-4. -   Schmidt, T. G. and A. Skerra (1993). “The random peptide     library-assisted engineering of a C-terminal affinity peptide,     useful for the detection and purification of a functional Ig Fv     fragment.” Protein Eng 6(1): 109-22. -   Schroeder, S. C., B. Schwer, et al. (2000). “Dynamic association of     capping enzymes with transcribing RNA polymerase II.” Genes Dev     14(19): 2435-2440. -   Schurer, H., K. Lang, et al. (2002). “A universal method to produce     in vitro transcripts with homogeneous 3′ ends.” Nucleic Acids Res     30(12): e56. -   Shao, W. H., X. E. Zhang, et al. (2000). “Anchor-chain molecular     system for orientation control in enzyme immobilization.” Bioconjug     Chem 11(6): 822-826. -   Spehner, D., S. Gillard, et al. (1988). “A cowpox virus gene     required for multiplication in Chinese hamster ovary cells.” J Virol     62(4): 1297-1304. -   Studier, F. W., A. H. Rosenberg, et al. (1990). “Use of T7 RNA     polymerase to direct expression of cloned genes.” Methods Enzymol     185: 60-89. -   Tang, Y., N. Jiang, et al. (1996). “Selection of linkers for a     catalytic single-chain antibody using phage display technology.” J     Biol Chem 271(26): 15682-15686. -   Ting, A. (2003). Genetically encoded fluorescent reporters of     kinase, methyltransferase, and acetyl-transferase activities. -   Ting, A. Y., K. H. Kain, et al. (2001). “Genetically encoded     fluorescent reporters of protein tyrosine kinase activities in     living cells.” Proc Natl Acad Sci USA 98(26): 15003-15008. -   Tiranti, V., A. Savoia, et al. (1997). “Identification of the gene     encoding the human mitochondrial RNA polymerase (h-mtRPOL) by     cyberscreening of the Expressed Sequence Tags database.” Hum Mol     Genet 6(4): 615-625. -   Tommasino, M., S. Ricci, et al. (1988). “Genome organization of the     killer plasmid pGK12 from Kluyveromyces lactis.” Nucleic Acids Res     16(13): 5863-5878. -   Topper, J. N. and D. A. Clayton (1989). “Identification of     transcriptional regulatory elements in human mitochondrial DNA by     linker substitution analysis.” Mol Cell Biol 9(3): 1200-1211. -   Turner, D. J., M. A. Ritter, et al. (1997). “Importance of the     linker in expression of single-chain Fv antibody fragments:     optimisation of peptide sequence using phage display technology.” J     Immunol Methods 205(1): 43-54. -   Ucker, D. S. and K. R. Yamamoto (1984). “Early events in the     stimulation of mammary tumor virus RNA synthesis by glucocorticoids.     Novel assays of transcription rates.” J Biol Chem 259(12):     7416-7420. -   Uptain, S. M. and M. J. Chamberlin (1997). “Escherichia coli RNA     polymerase terminates transcription efficiently at rho-independent     terminators on single-stranded DNA templates.” Proc Natl Acad Sci     USA 94(25): 13548-13553. -   Walker, S. C., J. M. Avis, et al. (2003). “General plasmids for     producing RNA in vitro transcripts with homogeneous ends.” Nucleic     Acids Res 31(15): e82. -   Wells, J. A. and D. B. Powers (1986). “In vivo formation and     stability of engineered disulfide bonds in subtilisin.” J Biol Chem     261(14): 6564-6570. -   Whitlow, M., B. A. Bell, et al. (1993). “An improved linker for     single-chain Fv with reduced aggregation and enhanced proteolytic     stability.” Protein Eng 6(8): 989-995. -   Wickham, T. J., M. E. Carrion, et al. (1995). “Targeting of     adenovirus penton base to new receptors through replacement of its     RGD motif with other receptor-specific peptide motifs.” Gene Ther     2(10): 750-756. -   Wu, S. C., J. C. Yeung, et al. (2002). “Design, production, and     characterization of an engineered biotin ligase (BirA) and its     application for affinity purification of staphylokinase produced     from Bacillus subtilis via secretion.” Protein Expr Purif 24(3):     357-65. -   Wyatt, L. S., B. Moss, et al. (1995). “Replication-deficient     vaccinia virus encoding bacteriophage T7 RNA polymerase for     transient gene expression in mammalian cells.” Virology 210(1):     202-205. -   Yue, Z., E. Maldonado, et al. (1997). “Mammalian capping enzyme     complements mutant Saccharomyces cerevisiae lacking mRNA     guanylyltransferase and selectively binds the elongating form of RNA     polymerase II.” Proc Natl Acad Sci USA 94(24): 12898-12903. -   Zabner, J., A. J. Fasbender, et al. (1995). “Cellular and molecular     barriers to gene transfer by a cationic lipid.” J Biol Chem 270(32):     18997-19007. -   Zhang, X. and F. W. Studier (1997). “Mechanism of inhibition of     bacteriophage T7 RNA polymerase by T7 lysozyme.” J Mol Biol 269(1):     10-27. 

The invention claimed is:
 1. A hetero-oligomeric enzyme comprising: a catalytic domain of a RNA triphosphatase; a catalytic domain of a guanylyltransferase; a catalytic domain of a N7-guanine methyltransferase; and a catalytic domain of a DNA-dependent RNA polymerase, wherein the catalytic domain of a DNA-dependent RNA polymerase is linked covalently or non-covalently with the catalytic domain of a RNA triphosphatase, the catalytic domain of a guanylyltransferase, or the catalytic domain of a N7-guanine methyltransferase, and wherein the hetero-oligomeric enzyme is non-natural.
 2. The hetero-oligomeric enzyme according to claim 1, wherein the catalytic domain of a N7-guanine methyltransferase and the catalytic domain of a DNA-dependent RNA polymerase are included in a monomer.
 3. The hetero-oligomeric enzyme according to claim 1, wherein the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase are included in a monomer.
 4. The hetero-oligomeric enzyme according to claim 1, wherein the catalytic domain of a RNA triphosphatase, the catalytic domain of a guanylyltransferase and the catalytic domain of a DNA-dependent RNA polymerase are included in a monomer.
 5. The hetero-oligomeric enzyme according to claim 1, further comprising a domain that enhances the activity of at least one catalytic domain of the hetero-oligomeric enzyme.
 6. The hetero-oligomeric enzyme according to claim 1, wherein at least one of the catalytic domain of a N7-guanine methyltransferase, the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase is a catalytic domain of the vaccinia mRNA capping enzyme.
 7. The hetero-oligomeric enzyme according to claim 1, comprising: a first monomer comprising a catalytic domain of a N7-guanine methyltransferase and a catalytic domain of a DNA-dependent RNA polymerase, and a second monomer comprising a catalytic domain of a RNA triphosphatase and a catalytic domain of a guanylyltransferase, wherein the catalytic domain of a N7-guanine methyltransferase is a catalytic domain of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase are catalytic domains of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and wherein the hetero-oligomeric enzyme further comprises a 31-kDa subunit encoded by the vaccinia virus D12L gene.
 8. The hetero-oligomeric enzyme according to claim 1, comprising: a first monomer comprising a catalytic domain of a N7-guanine methyltransferase, and a second monomer comprising a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, and a catalytic domain of a DNA-dependent RNA polymerase, wherein the catalytic domain of a N7-guanine methyltransferase is a catalytic domain of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and the catalytic domain of a RNA triphosphatase and the catalytic domain of a guanylyltransferase are catalytic domains of the vaccinia mRNA capping enzyme encoded by the vaccinia virus D1R gene, and wherein the hetero-oligomeric enzyme further comprises a 31-kDa subunit encoded by the vaccinia virus D12L gene.
 9. A method for producing an RNA molecule with a 5′-terminal m⁷GpppN cap, comprising contacting a DNA sequence encoding the RNA molecule with the hetero-oligomeric enzyme according to claim 1, wherein the DNA sequence is operatively linked to a promoter for the catalytic domain of a DNA-dependent RNA polymerase.
 10. A hetero-oligomeric enzyme comprising: a monomer comprising a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, and a catalytic domain of a N7-guanine methyltransferase; and a complex comprising a catalytic domain of a DNA-dependent RNA polymerase, wherein the monomer and the complex are linked covalently or non-covalently, and wherein the hetero-oligomeric enzyme is non-natural.
 11. The hetero-oligomeric enzyme according to claim 10, comprising a complex comprising a catalytic domain of a bacterial DNA-dependent RNA polymerase.
 12. The hetero-oligomeric enzyme according to claim 10, comprising a complex comprising a catalytic domain of the DNA-dependent RNA polymerase of Escherichia coli.
 13. A method for producing an RNA molecule with a 5′-terminal m⁷GpppN cap, comprising contacting a DNA sequence encoding the RNA molecule with the hetero-oligomeric enzyme according to claim 10, wherein the DNA sequence is operatively linked to a promoter for the catalytic domain of a DNA-dependent RNA polymerase. 